Treatment and prevention of immunodeficiency virus infection by administration of non-pyrogenic derivatives of lipid A

ABSTRACT

The present inventors have found that certain preparations containing LPS and/or lipid A variants, derivatives, and/or analogs demonstrate non-pyrogenic properties and exhibit anti-viral activities. In particular, non-pyrogenic preparations of LPS, lipid A, LPS antagonists and lipid A antagonists, and derivatives thereof induce β chemokine secretion, such as MIP-1β, but not proinflammatory cytokines, such as TNFα, IL-1β and IL-6. Non-pyrogenic preparations of the invention have been demonstrated by the Applicant to suppress HIV replication in human peripheral blood monocytes, as described by way of example herein. The present invention provides preparations of LPS or lipid A variants, analogs and derivatives of decreased or absent pyrogenicity which can be used as therapeutics for the treatment or prevention of immunodeficiency virus infection and its consequences.

This is a division of application Ser. No. 08/938,106, filed Sep. 26,2003 now U.S. Pat. No. 6,368,604.

1. FIELD OF THE INVENTION

The present invention relates to lipopolysaccharide (LPS) or lipid Avariants, derivatives, and analogs with non-pyrogenic and non-endotoxicproperties as well as methods for treatment and prevention ofimmunodeficiency virus infection, in particular HIV infection, usingthese LPS or lipid A variants and analogs and derivatives. The presentinvention also relates to LPS and lipid A antagonists and their use astherapeutics in the treatment and prevention of HIV infection. The LPSand lipid A variants, derivatives, and analogs of the present inventionpreferably induce the secretion of β chemokines but exhibit decreasedinduction relative to LPS and lipid A of secretion of proinflammatorycytokines, such as IL-1β, IL-6 and TNF-α. The present invention furtherrelates to pharmaceutical compositions for the treatment and preventionof HIV infection.

2. BACKGROUND OF THE INVENTION 2.1. The Human Immunodeficiency Virus

The human immunodeficiency virus (HIV) has been implicated as theprimary cause of the slowly degenerative immune system disease termedacquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F., et al.,1983, Science 220:868-870; Gallo, R., et al., 1984, Science224:500-503). There are at least two distinct types of HIV: HIV-1(Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., etal., 1984, Science 224:500-503) and HIV-2 (Clavel, F., et al., 1986,Science 233:343-346; Guyader, M., et al., 1987, Nature 326:662-669).Further, a large amount of genetic heterogeneity exists withinpopulations of each of these types. In humans, HIV replication occursprominently in CD4⁺ T lymphocyte populations, and HIV infection leads todepletion of this cell type and eventually to immune incompetence,opportunistic infections, neurological dysfunctions, neoplastic growth,and ultimately death.

HIV is a member of the lentivirus family of retroviruses (Teich, N., etal., 1984, RNA Tumor Viruses, Weiss, R., et al., eds., CSH-Press, pp.949-956). Retroviruses are small enveloped viruses that contain asingle-stranded RNA genome, and replicate via a DNA intermediateproduced by a virally-encoded reverse transcriptase, an RNA-dependentDNA polymerase (Varmus, H., 1988, Science 240:1427-1439). The HIV viralparticle comprises a viral core, composed in part of capsid proteins,together with the viral RNA genome and those enzymes required for earlyreplicative events. Myristylated gag protein forms an outer shell aroundthe viral core, which is, in turn, surrounded by a lipid membraneenvelope derived from the infected cell membrane. The HIV envelopesurface glycoproteins are synthesized as a single 160 kilodaltonprecursor protein which is cleaved by a cellular protease during viralbudding into two glycoproteins, gp41 and gp120. gp41is a transmembraneglycoprotein and gp120 is an extracellular glycoprotein which remainsnon-covalently associated with gp41, possibly in a trimeric ormultimeric form (Hammarskjold, M., & Rekosh, D., 1989, Biochem. Biophys.Acta 989:269-280).

HIV is targeted to CD4⁺ cells because a CD4 cell surface protein (CD4)acts as the cellular receptor for the HIV-1 virus (Dalgleish, A., etal., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry intocells is dependent upon gp120 binding the cellular CD4 receptormolecules (McDougal, J. S., et al., 1986, Science 231:382-385; Maddon,P. J., et al., 1986, Cell 47:333-348), explaining HIV's tropism for CD4⁺cells, while gp41 anchors the envelope glycoprotein complex in the viralmembrane. While these virus:cell interactions are necessary forinfection, there is evidence that additional virus:cell interactions arealso required.

2.2. HIV Treatment

HIV infection is pandemic and HIV-associated diseases represent a majorworld health problem. Although considerable effort is being put into thedesign of effective therapeutics, currently no curative anti-retroviraldrugs against AIDS exist. In attempts to develop such drugs, severalstages of the HIV life cycle have been considered as targets fortherapeutic intervention (Mitsuya, H., et al., 1991, FASEB J.5:2369-2381). Many viral targets for intervention with HIV life cyclehave been suggested, as the prevailing view is that interference with ahost cell protein would have deleterious side effects. For example,virally encoded reverse transcriptase has been one focus of drugdevelopment. A number of reverse-transcriptase-targeted drugs, including2′,3′-dideoxynucleoside analogs such as AZT, ddI, ddC, and d4T have beendeveloped which have been shown to been active against HIV (Mitsuya, H.,et al., 1991, Science 249:1533-1544).

The new treatment regimens for HIV-1 show that a combination of anti-HIVcompounds, which target reverse transcriptase (RT), such asazidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddI),dideoxycytidine (ddC) used in combination with an HIV-1 proteaseinhibitor have a far greater effect (2 to 3 logs reduction) on viralload compared to AZT alone (about 1 log reduction). For example,impressive results have recently been obtained with a combination ofAZT, ddI, 3TC and ritonavir (Perelson, A. S., et al., 1996, Science15:1582-1586). However, it is likely that long-term use of combinationsof these chemicals will lead to toxicity, especially to the bone marrow.Long-term cytotoxic therapy may also lead to suppression of CD8⁺ Tcells, which are essential to the control of HIV, via killer cellactivity (Blazevic, V., et al., 1995, AIDS Res. Hum. Retroviruses11:1335-1342) and by the release of suppressive factors, notably thechemokines Rantes, MIP-1a and MIP-1β (Cocchi, F., et al., 1995, Science270:1811-1815). Another major concern in long-term chemicalanti-retroviral therapy is the development of HIV mutations with partialor complete resistance (Lange, J. M., 1995, AIDS Res. Hum. Retroviruses10:S77-82). It is thought that such mutations may be an inevitableconsequence of anti-viral therapy. The pattern of disappearance ofwild-type virus and appearance of mutant virus due to treatment,combined with coincidental decline in CD4⁺ T cell numbers stronglysuggests that, at least with some compounds, the appearance of viralmutants is a major underlying factor in the failure of AIDS therapy.

Attempts are also being made to develop drugs which can inhibit viralentry into the cell, the earliest stage of HIV infection. Here, thefocus has thus far been on CD4, the cell surface receptor for HIV.Recombinant soluble CD4, for example, has been shown to inhibitinfection of CD4⁺ T cells by some HIV-1 strains (Smith, D. H., et al.,1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however,are relatively less sensitive to inhibition by recombinant CD4 (Daar,E., et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition,recombinant soluble CD4 clinical trials have produced inconclusiveresults (Schooley, R., et al., 1990, Ann. Int. Med. 112:247-253; Kahn,J. O., et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R., et al.,1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).

The late stages of HIV replication, which involve crucial virus-specificprocessing of certain viral encoded proteins, have also been suggestedas possible anti-HIV drug targets. Late stage processing is dependent onthe activity of a viral protease, and drugs are being developed whichinhibit this protease (Erickson, J., 1990, Science 249:527-533).

Recently, chemokines produced by CD8⁺ T cells have been implicated insuppression of HIV infection (Paul, W. E., 1994, Cell 82:177; Bolognesi,D. P., 1993, Semin. Immunol. 5:203). The chemokines RANTES, MIP-1α andMIP-1β, which are secreted by CD8⁺ T cells, were shown to suppress HIV-1p24 antigen production in cells infected with HIV-1 or HIV-2 isolates invitro (Cocchi, F, et al., 1995, Science 270:1811-1815). Thesechemokines, alone or in combination, effectively suppressed thereplication of several primary isolates of HIV-1, HIV-2 and SIV whentested in a variety of in vitro assays (Cocchi et al. supra). Themechanism of chemokine-mediated suppression was further delineated by aseries of independent reports showing that β chemokine suppression CCR5serves as a co-receptor for macrophage-tropic NSI isolates of HIV(Alkhatib et al., 1996, Science 272:1955; Dragic et al., 1996, Nature381:667; Choe et al., 1996, Cell 85:1135; Berson et al., 1996, J.Virology 70:6288). However, this activity is highly specific since βchemokines blocked macrophage tropic NSI isolates but had no significanteffect on T cell-tropic SI isolates of HIV-1 (Cocchi et al., supra;Alkhatib et al., supra). Thus, these and other chemokines may proveuseful in therapies for some strains of HIV infection. The clinicaloutcome, however, of all these and other candidate drugs is still inquestion.

Attention is also being given to the development of vaccines for thetreatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120,gp41) have been shown to be the major antigens for anti-HIV antibodiespresent in AIDS patients (Barin et al., 1985, Science 228:1094-1096).Thus far, therefore, these proteins seem to be the most promisingcandidates to act as antigens for anti-HIV vaccine development. Severalgroups have begun to use various portions of gp160, gp120, and/or gp41as immunogenic targets for the host immune system. See for example,Ivanoff, L., et al., U.S. Pat. No. 5,141,867; Saith, G., et al., PCTInternational Publication No. WO92/22,654; Shafferman, A., PCTInternational Publication No. WO91/09,872; Formoso, C., et al., PCTInternational Publication No. WO90/07,119. To this end, vaccinesdirected against HIV proteins are problematic in that the virus mutatesrapidly rendering many of these vaccines ineffective. Clinical resultsconcerning these candidate vaccines, however, still remain far in thefuture.

Thus, although a great deal of effort is being directed to the designand testing of anti-retroviral drugs, effective, non-toxic treatmentsare still needed.

2.3. Lipopolysaccharides

Endotoxins of gram-negative microorganisms fulfill a vital function forbacterial viability, and induce in mammalians potent pathophysiologicaleffects. Chemically, they are lipopolysaccharides consisting of anO-specific chain, a core oligosaccharide, and a lipid component, termedlipid A. The latter determines the endotoxic activities and togetherwith the core constituent 3-deoxy-D-manno-octulosonic acid (KDO),essential functions for bacteria.

Under normal conditions, lipopolysaccharide (LPS) is inserted in theouter surface of the outer membrane of gram negative bacteria(Schnaitman and Klena, Microbiol Rev, 57:655-682 (1993)); and Makela andStocker, In: Handbook of endotoxin volume 1, Elsevier Biomedical Press,Amsterdam, Rietschel (ed), pp. 59-137 (1984). Complete or “smooth” LPSis composed of three main domains called lipid A, the O-antigen (alsocalled the O-polysaccharide) and the core region, which creates anoligosaccharide link between lipid A and the O antigen (Schnaitman andKlena, supra; and Makela and Stocker, supra). The O-antigen is composedof oligosaccharide repeat units. The structure and number of theserepeats varies depending on the bacterial species and growth conditions,typically ranging from one to fifty repeats (Schnaitman and Klena,supra; and Makela and Stocker, supra). Some bacterial generi, such asNeisseria spp., produce LPS that has low numbers of O-antigen repeatsand therefore is referred to as lipoligosaccharide (LOS) simply toreflect this fact (Schnaitman and Klena, supra; and Makela and Stocker,supra).

The biological properties of LPS have been extensively investigated(Rietschel et al, FASEB J, 8:217-225 (1994); and Raetz, J Bacteriol,175:5745-5753 (1993)). This molecule has powerful pyrogenic propertiesand in humans purified LPS (at doses of 200 ng to 1 μg) has been shownto induce febrile responses (Greisman and Homick, J Immunol,109:1210-1215 (1972); Greisman and Homick, J Infect Dis, 128:257-263(1973); Abemathy and Spink, J Clin Invest, 37:219-225 (1958); Rietschelet al, supra; and Raetz, supra (1993)). These febrile responses aremediated by host proinflammatory cytokines IL-1, IL-6, and TNF-α, thesecretion of which is induced by LPS (Rietschel et al, supra and Raetz,supra).

The biologically active component of LPS is lipid A (Rietschel et al,supra; Verma et al, Infect Immun, 60(6):2438-2444 (1992); Alving, JImmunol Meth, 140:1-13 (1991); Alving and Richards, Immunol Lett,25:275-280 (1990); and Richard et al, Infect Immun, 56:682-686 (1988)).Activity analysis of lipid A biosynthesis precursors or syntheticintermediates showed that various elements of lipid A are essential forpyrogenicity (Rietschel et al, supra; Raetz, supra).

For several years, it has been known that under certain circumstances,stimulation with bacterial LPS protects macrophages from HIV infection(Kornbluth et al., 1989, J. Exp. Medicine 169:1137; Bernstein et al.,1991, J. Clinical Invest. 88:540; Bagasra et al., 1992, Proc. Natl.Acad. Sci. 89:6285). LPS-mediated suppression is thought to be dependenton LPS-CD14 interactions (Bagasra et al. supra), the induction of βchemokines MIP-1α, MIP-1β and RANTES (Verani et al., 1997, J. Exp. Med.185:805) and down regulation of β chemokine receptors (Sica et al, 1997,J. Exp. Med. 185:969).

In attempts to detoxify the effects of LPS or lipid A in the treatmentof Gram-negative bacteremia and septic shock, antibodies have beendesigned which detoxify the endotoxin activity by hydrolyzing the LPS orlipid A to products with reduced toxicity (see U.S. Pat. No. 5,597,573,issued Jan. 28, 1997).

LPS and lipid A are potent activators of proinflammatory cytokines,which accounts for the pyrogenic nature of these molecules. LPS has beenshown to suppress HIV replication. LPS-induced suppression of HIV may bemediated through induction and/or down regulation of chemokinereceptors. However, due to the toxicity of this molecule, LPS and lipidA are not viable candidates for the treatment of HIV.

Citation of references hereinabove shall not be construed as anadmission that such references are prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present inventors have found that certain preparations containingLPS and/or lipid A variants, derivatives, and/or analogs demonstratenon-pyrogenic properties and exhibit anti-viral activities, particularlyanti-HIV activities. In particular, non-pyrogenic preparations of LPS,lipid A, LPS antagonists and lipid A antagonists, and derivativesthereof induce β chemokine secretion, such as MIP-1α and MIP-1β, but notproinflammatory cytokines, such as TNFα, IL-1β and IL-6. Thenon-pyrogenic preparations of the invention, have been demonstrated bythe Applicant to suppress HIV replication in human peripheral bloodmonocytes, as described by way of example herein. The present inventionprovides preparations of reduced or substantially negligiblepyrogenicity of LPS variants and lipid A variants, and analogs andderivatives thereof which may be used as therapeutics for the treatmentof human immunodeficiency virus infection.

The present invention also encompasses synthetic lipid A and LPSantagonists, such as, but not limited to, lipid X and lipid IV_(A),which suppress immunodeficiency virus replication, in particular, HIV-1replication, and exhibit decreased induction relative to LPS and lipid Aof proinflammatory cytokines such as IL-6, TNFα and IL-1β. Thelipopolysaccharide compositions of the present invention include thoseantagonists, derivatives or analogs of LPS and lipid A which exhibitreduced pyrogenicity and proinflammatory activity relative to wild-typeLPS and lipid A, respectively, yet stimulate β chemokine secretion andinhibit HIV replication. The present invention fills a tremendous needfor a non-toxic, long-term treatment of HIV infection and its sequelae,ARC and AIDS.

In particular, the present invention relates to LPS or lipid Apreparations isolated from gram negative organisms containing at leastone mutation from the group kdsA, kdsB, htrB, msbB. In a preferredembodiment of the present invention, non-pyrogenic LPS is isolated fromthe E. coli htrB1::Tn10 msbB::Ωcam double mutant MLK986.

The present invention further relates to preparations of LPS or lipid Awhich have been differentially modified to yield reduced pyrogenicityor, preferably, substantially non-pyrogenic properties of thepreparation relative to wild-type LPS and lipid A, respectively. Inspecific embodiments, preparations are treated by alkaline hydrolysis oracyloxyacyl hydrolase. Modified derivatives also in accordance with thepresent invention are derived from the group of monophosphoryl lipid A,penta-acyl lipid A, lipid IV_(A) or lipid X. The present invention stillfurther relates to LPS or lipid A derived from deacylation, by treatmentwith an alkali.

The present invention further relates to therapeutic methods andcompositions for treatment and prevention of diseases and disordersassociated with HIV-1 infection based on LPS or lipid A derivatives andtherapeutically and prophylactically effective preparations containing aderivative of LPS or lipid A, and related analogs. Non-pyrogenicderivatives of lipid A and LPS can be identified by their failure toelicit a toxic response in mammals, their lack of proinflammatoryactivity, and/or their lack of induction of secretion of significantlevels of pyrogenic cytokines, including IL-1β, IL-6 and TNFα.Preferably, non-pyrogenic derivatives are used in the therapeuticmethods and compositions of the invention; alternatively, derivatives ofreduced pyrogenicity relative to wild-type LPS and lipid A may beemployed.

The invention provides for the treatment and prevention of HIV infectionby administration of a therapeutic compound of the invention. Thetherapeutic compounds of the present invention include: lipid A or LPSderived from gram negative organisms containing at least one mutationselected from the group kdsA, kdsB, htrB, msbB, and derivatives andanalogs of he foregoing; preparations of lipid A or LPS which have beenmodified to have reduced pyrogenic properties, including but not limitedto, the group of monophosphoryl lipid A, penta-acyl lipid A or lipid Aor LPS derivatives derived by deacylation of lipid A or LPS, treatmentof LPS and lipid A with acyloxyacl hydroxylase or by treatment with analkali, and derivatives and analogs of the foregoing. The invention alsoprovides in vitro and in vivo assays for assessing the efficacy oftherapeutics of the invention for treatment or prevention of HIV. Theinvention also provides pharmaceutical compositions and methods ofadministration of therapeutics of the invention for treatment orprevention of HIV infection.

3.1. Definitions

As used herein, the following terms shall have the meanings indicated.

AIDS Acquired Immune Deficiency Syndrome ARC AIDS-Related Complex KSKaposi's Sarcoma OI Opportunistic Infection PBMC Peripheral BloodMononuclear Cell

4. DESCRIPTION OF THE FIGURES

FIG. 1. Proinflammatory Activity of W3110 LPS and Mutant DerivativeMLK986 LPS. LPS was extracted from E. coli strain W3110 and ahtrB1::Tn10, msbB::Ωcam double mutant (MLK986) after being cultured at a30° C., 37° C. or 42° C. To characterize the proinflammatory activity ofLPS harvested from W3110 and MLK986, the level of TNFα in culturesupernatants was measured 8 hrs after stimulation of human PBMCs.Significant levels of TNFα secretion was observed by the parent strainW3110 LPS at concentrations above 1 mg/ml irrespective of culturetemperature. In contrast, LPS from mutant strain MLK986 cultured at 30°C. only moderately elicited TNFα secretion and LPS derived from MLK986cultured at 37° C. and 42° C. even at concentrations as high as 1 μg/mldid not elicit TNFα secretion in the human PBMC activation assay. FigureLegend:

-   -   W/42    -   W/37    -   W/30    -   MLK/42    -   MLK/37    -   MLK/30

FIG. 2. LPS From MLK986 Stimulates β Chemokine Secretion. LPS isolatedfrom MLK986, cultured at either 37° C. or 42° C., and RsDPLA stimulatedthe secretion of MIP-1α and MIP-1β from human PBMCs in a dose-dependentmanner. Figure Legend:

-   -   MLK986/37    -   MLK986/42    -   RsDPLA

FIG. 3. The Peak Production of Chemokines Occurred 24 Hours After MLK986Stimulation. The peak stimulation of chemokine production from humanPBMCs by LPS isolated from MLK986, cultured at either 37° C. or 42° C.,and RsDPLA occurred at 24 hours. The level of RANTES was notsignificantly elevated after stimulation with MLK986 LPS or RsDPLA.Figure Legend:

-   -   MLK986/37    -   MLK986/42    -   RsDPLA    -   Unstimulated

FIGS. 4A-C. Inhibition of HIV-1 Replication by Non-pyrogenic Forms ofLPS. FIG. 4A is representative of 4 different experiments anddemonstrates that MLK986/37 inhibits HIV-1 chemokines. FIG. 4B MLK986/37induced HIV-1 inhibition in MDM is reversed by addition of a mixture ofneutralizing antibodies against RANTES, MIP-1α, and MIP-1β (from R&DSystems Inc.; 200 ug/ml each). FIG. 4C HIV-1 replication inhibitionoccurred without inducing pyrogenic cytokines. Figure Legend:

-   -   Control    -   LPS    -   LPS mutant

FIG. 5. Lipid A antagonist, lipid IV_(A), inhibits HIV replication.PBMCs were treated with synthetic lipid IV_(A) at 1000 ng/ml, 100 ng/mland 10 ng/ml for 24 hours. The treated cells were subsequently infectedwith 0.200 ng of HIV-1_(BAL) (Cocchi et al, supra) for 2 hours, washedtwice and incubated for a further 19 days. Culture supernatants werethen collected and the level of p24 in these supernatants was measuredby ELISA (R&D Systems). The results of this assay show that lipid IV_(A)suppresses the replication of HIV-1_(BAL) in a dose-dependent manner.Figure Legend:

-   -   Lipid IVa (1000 ng/ml)    -   Lipid IVa (100 ng/ml)    -   Lipid IVa (10 ng/ml)    -   MLK986/42 (1000 ng/ml)    -   MLK986/42 (100ng/ml)    -   MLK986/42 (10 ng/ml)

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to forms of lipid A and LPS which havereduced pyrogenicity as exhibited by reduced proinflammatory andendotoxic activity, relative to wild-type lipid A and LPS respectively,yet stimulate secretion of β-chemokines and are effective at inhibitingHIV replication and/or infection in vitro or in vivo, decreasing viralload, and/or treating or preventing disorders associated with HIVinfection. The present invention also relates to LPS and lipid Aantagonists which have reduced proinflammatory activity, yet stimulatesecretion of β-chemokines and are effective at inhibiting HIVreplication and/or infection in vitro or in vivo, decreasing viral load,and/or treating or preventing disorders associated with HIV infection.The LPS and lipid A analogs (including antagonists) of the presentinvention preferably induce the secretion of β-chemokines but exhibitdecreased induction relative to wild-type LPS and lipid A of thesecretion of proinflammatory cytokines, such as, IL-β, IL-6 and TNF-α,thereby providing a non-toxic treatment of immunodeficiency virusinfection, in particular, HIV infection and its sequelae, ARC and AIDS.The LPS and lipid A variants, derivatives, and analogs of the presentinvention exhibit decreased pyrogenicity i.e., preferably induce thesecretion of β chemokines but exhibit substantial induction relative towild-type LPS and lipid A of proinflammatory cytokines. Non-pyrogenicderivatives of lipid A and LPS can be identified by their failure toelicit a toxic or endotoxic response in mammals, their lack ofproinflammatory activity and/or their lack of induction of secretion ofsignificant levels of pyrogenic cytokines, e.g., IL-7B, IL-6, TNFα.Preferably, non-pyrogenic derivatives are used in the therapeuticmethods and compositions of the invention; alternatively, derivatives ofreduced pyrogenicity relative to wild-type LPS or lipid A may beemployed. LPS antagonists and lipid A antagonists can be identified bytheir ability to interfere or compete with the activities of LPS orlipid A, e.g., to competitively inhibit the interaction between LPS andthe cellular receptor for LPS. Thus, by way of example, such anantagonist may be identified by its ability to reduce the physiologicalmanifestation of LPS or lipid A activity, e.g., its ability to reduceTNFα secretion from LPS-stimulated PBMCs.

Efficacy in treating or preventing HIV infection may be demonstrated bydetecting the ability to inhibit the replication of the HIV virus, toinhibit HIV transmission, or to prevent HIV from establishing itself inits host, or to prevent, ameliorate or alleviate the symptoms of adisease caused by HIV infection, or prevent disease progression. Thetreatment is considered therapeutic if there is, for example, areduction in viral load, decrease in mortality and/or morbidity.

In specific embodiments, the invention provides an LPS variant withsubstantially reduced pyrogenicity isolated from gram negative organismscontaining at least one mutation selected from the group kdsA, kdsB,htrB or msbB. The present invention further provides an isolatedpreparation of LPS which has been modified relative to wild-type toyield its reduced or non-pyrogenic properties, including but not limitedto the group of monophosphoryl lipid A, penta-acyl lipid A, lipid IV_(A)or lipid X. The present invention further provides for analogs orderivatives of lipid A or lipopolysaccharide achieved by deacylation, bythe treatment with acyloxyacyl hydroxylase or by the treatment with analkali. The present invention further provides synthetic LPS and lipid Amolecules which substantially lack or exhibit reduced proinflammatoryactivity and are effective at inhibiting HIV replication.

The present invention further relates to therapeutic methods andcompositions for treatment and prevention of disorders associated withimmunodeficiency virus infection based on non-pyrogenic orreduced-pyrogenic LPS and lipid A preparations and therapeuticallyeffective analogs and derivatives thereof. The invention provides fortreatment of HIV infection by administration of a therapeutic compoundof the invention. The therapeutic compounds of the invention includepreparations of reduced or substantially absent pyrogenicity of LPS andlipid A which do induce β chemokines, such as MIP-1α and MIP-1β, andrelated derivatives and analogs thereof. Lipopolysaccharides and lipid Avariants, analogs and derivatives which are effective for treatment andprevention of HIV infection can be identified by in vitro and in vivoassays such as those described in Section 5.3 infra.

In a preferred embodiment, a therapeutic composition of the inventioncomprises an isolated lipopolysaccharide isolated from gram negativeorganisms containing at least one mutation from the group kdsA, kdsB,htrB or msbB, or a synthetic analog of lipopolysaccharide which has beenmodified relative to wild-type to yield reduced or absent pyrogenicproperties, including but not limited to the group of monophosphoryllipid A, penta-acyl lipid A, lipid IV_(A) or lipid X. In other preferredembodiments, the therapeutic comprises a lipopolysaccharide analogue orderivative achieved by deacylation, by treatment with acyloxyacylhydroxylase or by treatment with an alkali. In yet another preferredembodiment, the therapeutic comprises synthetic LPS and lipid Amolecules which lack or exhibit reduced proinflammatory activity and areeffective at inhibiting HIV replication.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections withfollow.

5.1. Lipopolysacchride Variants and Derivatives and Analogs Thereof withReduced or Absent Pyrogenicity

The invention provides isolated preparations of non-pyrogenic or reducedpyrogenic variants of LPS and lipid A and isolated preparations of LPSor lipid A antagonists which exhibit decreased induction relative towild-type LPS and lipid A of proinflammatory activity, yet preferablystimulate secretion of β chemokines and are effective for treatment orprevention of immunodeficiency virus and/or HIV infection and resultingdisorders. Pyrogenicity of the preparations may be determined bymeasuring the ability of the LPS or lipid A preparation to stimulate thesecretion of proinflammatory cytokines, e.g., IL-β, IL-6 and TNFα, or byany method known in the art. Effectiveness of the lipopolysaccharides ofthe invention for treatment or prevention of HIV infection can bedetermined by any of the methods disclosed in Section 5.3 infra or byany method known in the art. In a specific embodiment, the LPS and lipidA preparations and derivatives or analogs thereof inhibit HIV infection.

In a preferred embodiment the lipid A antagonists or lipopolysaccharidesare derived from gram negative microorganisms that have mutations in oneof the following genes: kdsA, kdsB, htrB or msbB.

In a preferred embodiment of the present invention, lipid X or lipidIV_(A), both LPS and lipid A antagonists of reduced or absentpyrogenicity, are used for the treatment of HIV. Lipid X is amonosaccharide precursor of lipid A (Rietschel et al., supra; and Raetz,supra (1993) and lipid IV_(A), a tetra-acyl precursor of lipid A (Wanget al., Infect Immun, 59(12):4655-4664. (1991); Ulmer et al., InfectImmun., 60(12):145-5152 (1992); Kovach et al., J. Exp. Med., 172:77-84(1990) and Rietschel et al., supra). These molecules are of interestbecause both lipid X and lipid IV_(A) display non-pyrogeniccharacteristics in vitro (Golenbock et al., Infect. Immun., 56:779(1988); Golenbock et al., J. Biol. Chem., 266:19490 (1991); Wang et al.,Infect. Immun., 59(12); 4655-4664 (1991); Ulmer et al., Infect Immun.,60(12):145-5152 (1992); Kovach et al., J. Exp. Med., 172:77-84 (1990);Rietschel et al., supra; and Raetz, supra). In addition, lipid X andlipid IV_(A) are LPS and lipid A antagonists (Wang et al., supra; Ulmeret al., supra; Kovach et al., supra; Rietschel et al., supra; and Raetz,supra). The activity of these molecules differs, since lipid X isnon-pyrogenic in mice, whereas lipid IV_(A) displays similar toxicity ofthat of lipid A in mice (Golenbock et al., supra (1988); Golenbock etal., supra (1991)). Furthermore, CD14 has been shown to enhance thecellular responses to LPS and lipid IV_(A) in mice without impartingligand-specific recognition (Delude et al., Proc. Natl. Acad. Sci.,92:9288 (1995)). Together, these results suggested that individual LPSantagonists may operate through distinct mechanisms.

In yet another preferred embodiment of the present invention, forms ofLPS of reduced or absent pyrogenicity isolated from mutant strains ofgram negative bacteria may be used in accordance with the presentinvention to treat HIV. There is growing evidence that mutations in htrband msbB may influence the biosynthesis of lipid A. These mutants aretemperature sensitive and LPS isolated from these mutants stains lessintensely on silver-stain gels (Karow et al., J. Bacteriol, 173:741-750(1991); Karow and Georgopoulos, J. Bacteriol 174:702-710 (1992)).Although the basis for the temperature-sensitive growth phenotype of thehtrb and msbB mutants has remained cryptic, there has been speculationthat these mutants produce defective lipid A precursors (Karow andGeorgopoulos, supra). This assumption was based on altered membranelipid content (Karow et al., J. Bacteriol., 174:7407 (1992)). E. colimutants carrying mutations in htrb, msbB or both htrb and msbB producenon-pyrogenic LPS when grown at temperatures above 33° C. and below 44°C. (PCT International Publication No. WO 97/18837 dated May 29, 1997).This non-pyrogenic LPS from E. coli mutants carrying mutations in htrb,msbB or both htrB and msbB also displays LPS antagonist activity (PCTInternational Publication No. WO 97/18837). Recent evidence demonstratedthat HtrB and MsbB function as myristoyl and lauroyl transferases,respectively and are necessary for the synthesis of complete lipid A(Clementz et al., J. Biol. Chem., 271:12095 (1996); Summerville et al.,J. Clin. Invest., 97:359 (1996). Collectively, these data suggest thatthe non-pyrogenic or reduced-pyrogenic property of LPS isolated from E.coli strains carrying mutations in htrB, msbB or both htrB and msbB whengrown at temperatures above 33° C. and below 44° C. is the result of anaccumulation of lipid A precursors, such as lipid IV_(A) (in htrB,mutants) and penta-acyl lipid A (in the single msbB mutant).

Thus, several mutants of E. coli with defective lipid A biosynthesishave been shown to accumulate LPS and lipid A analogs with LPSantagonist activity and LPS analogs isolated from these strains may beused in accordance with the present invention to treat or prevent HIVinfections and disorders associated therewith.

In another embodiment of the present invention, LPS antagonists to beused in accordance with the present invention may be produced bydeacylation of LPS or lipid A. LPS antagonists may be developed usingchemical (Neter et al., J. Immunol., 76:377 (1956); Qureshi et al., J.Biol. Chem., 266:6532 (1991)) or enzymatic (Munford and Hall, J. Biol.Chem., 264:15613 (1989); Erwin and Munford, J. Biol. Chem., 256:16444(1990)) procedures known to those of ordinary skill in the art. Chemicalproduction of LPS antagonists from LPS or lipid A can be accomplished byalkaline hydrolysis of LPS or lipid A (Neter et al., supra; Qureshi etal., supra). Enzymatic production of LPS antagonists from LPS or lipid Acan be accomplished by treating LPS or lipid A with acyloxyacylhydrolase. A source of acyloxyacl hydrolase are professional phagocyteswhich normally produce this enzyme to be utilized by the host todetoxify LPS (Munford and Hall, supra; Erwin and Munford, supra).

In another embodiment, LPS and lipid A compositions of reduced or absentpyrogenicity which may be used in accordance with the present inventionmay be isolated from bacteria such as Rhodobacter sphaeroides that haveLPS with reduced pyrogenicity. Not all gram negative bacteria produceLPS structures that display endotoxin activity (Salimath et al., Qureshiet al., J. Biol. Chem., 266:6532 (1991); Qureshi et al., J. Biol. Chem.,263:5502 (1988)). LPS structures which have significantly lesspyrogenicity than LPS isolated from E. coli, e.g., if the LPS structurestimulates significantly less or no secretion of IL-1β, IL-6 or TNFαfrom peripheral blood monocytes (PBMCs), the LPS structure may be usedin accordance with the present invention. For example, but not by way oflimitation, Rhodobacter sphaeroides LPS has an unusual pentaacylstructure that is significantly less pyrogenic that E. coli LPS in vitroand in vivo (Salimath et al., Eur. J. Biochem., 136:195 (1983); Qureshiet al., J. Biol. Chem., 266:6532 (1991). Monophosphoryl or diphosphorylRSLA can also be used in accordance with the present invention.Moreover, R. sphaeroides lipid A (RSLA) is an effective LPS antagonist(Salimath et al., Eur. J. Biochem., 136:195 (1983); Qureshi et al., J.Biol. Chem., 266:6532 (1991); Qureshi et al., J. Biol. Chem., 263:5502(1988)) and prevents LPS induced lethality in mice (Salimath et al.,Eur. J. Biochem., 136:195 (1983); Qureshi et al., J. Biol. Chem.,266:6532 (1991); Qureshi et al., J. Biol. Chem., 263:5502 (1988)). In afurther embodiment of the present invention, monophosphoryl RSLA may bea more effective LPS antagonist than diphosphoryl RSLA and therefore isused in accordance with an embodiment of the present invention for thetreatment of HIV (Salimath et al., Eur. J. Biochem., 136:195 (1983);Qureshi et al., J. Biol. Chem., 266:6532 (1991); Qureshi et al., Qureshiet al., J. Biol. Chem., 263:5502 (1988)).

In accordance with the present invention, lipopolysacchrides and lipid Aanalogs of reduced or absent pyrogenicity may be yielded by themodification of naturally occurring lipopolysaccharides. Suchmodification can include, but are not limited to treating thelipopolysaccharide or lipid A with acyloxyacyl hydrolase, or by aalkaline hydrolysis process or a deacylation process.

In yet a further embodiment of the present invention, synthetic LPSantagonists may be used in accordance with the present invention for thetreatment of HIV. For example, but not by way of limitation, syntheticlipid A and lipid X analogs (Ulmer et al., Infect. Immun, 60:5145(1992); Perera et al., Infect Immun., 61:2015 (1993); Wang et al.,Infect. Immun., 59:4655 (1991); Kotani et al., Infect. Immuno., 54:673(1986): Kotani et al., Infect Immun., 49:225 (1985); Fagan et al., J.Immunol., 153:5230 (1994)) can be used. Disaccharide LPS antagonists,which resemble lipid IV_(A), may also be used in accordance with thepresent invention (Ulmer et al., supra; Perera et al., supra); Wang etal., supra; Kotani et al., supra (1985); Kotani et al., supra (1986)). Asecond group of synthetic molecules were designed to be analogs of R.sphaeroides lipid A and are potent LPS antagonists (Christ et al.,Science 269:80 (1995); Christ et al., J. Am. Chem. Soc., 116:3637(1994)) and can be used in accordance with the present invention. Afurther set of synthetic LPS antagonists have been described that arecomposed of monosaccharide backbones and more closely resemble lipid X;these can be used in accordance with the present invention (Perera etal., supra). Collectively, these molecules have proven to display potentLPS antagonist activity in vitro (Ulmer et al., supra; Perera et al.,supra); Wang et al., supra; Kotani et al., supra (1985); Kotani et al.,supra (1986)) and have been evaluated for safety in humans (Bunnell etal., Crit. Care Med. Suppl., 23:147 (1995); Bunnell et al., Crit. CareMed. Suppl., 23: A151 (1995)). In a preferred embodiment, syntheticanalogs of LPS can be generated which contain a 2-deoxy-2-aminogluconateresidue in place of the glucosamine-1-phosphate at the reducing end andfurther bear a galacturonic acid moiety instead of a phosphate atposition 4′.

In another embodiment of the present invention, the lipopolysaccharidesand lipid A molecules and analogs and derivatives thereof can bemodified in accordance with the present invention so that they areshortened or condensed, e.g., the carbon backbone may be shortened to a5 carbon backbone. Further, the LPS and lipid A structures of thepresent invention may be modified so that one or more or all of theglucosamine residues are substituted with galactosamine residues. Thediphosphoryl LPS and lipid A structures of the present invention can beconverted to either nonphosphoryl or monophosphoryl LPS and lipid Astructures in accordance with the present invention. The lipid A and LPSstructures of the present invention can be modified to have more or lesscharge, e.g., the lipid A or LPS structures can be modified to be morecharged by the addition of amine groups. The lipid A and LPS structurescan further be modified so that they are less immunogenic, i.e., lessrecognized by the immune system of the host. In accordance with thepresent invention, any modification of the LPS and lipid A structures ofthe present invention which results in a non-pyrogenic orreduced-pyrogenic analog or derivative, i.e., analogs which exhibitdecreased induction relative to wild-type LPS and lipid A ofproinflammatory activity yet stimulate secretion of β chemokines and areeffective for treatment or prevention of HIV infection can be used. Theinvention thus also provides a method of screening LPS and -lipid Aderivatives and analogs for anti-immunodeficiency virus activity, e.g.,by assaying them for the ability to inhibit immunodeficiency virusreplication or expression of immunodeficiency virus RNA or protein or toalleviate symptoms of an immunodeficiency virus-induced disorder.

In another embodiment of the present invention, a mixture of one lipid Aor LPS or analog or derivative of the present invention mixed with atleast one other LPS or lipid A structure or analog or derivative thereofof the present invention can be used to treat or prevent HIV infectionsand disorders associated therewith. In accordance with a specificembodiment of the present invention, a mixture comprising at least oneLPS antagonist with an LPS or lipid A structure isolated from a gramnegative organism, wherein the LPS antagonist is in molar excess of theLPS or lipid A structure can be used to treat or prevent HIV infectionand disorders associated therewith.

The lipopolysaccharides and lipid A molecules and analogs andderivatives thereof of the present invention may be associated orconjugated with other molecules. These molecules may be macromolecularcarrier groups including, but not limited to, lipid-fatty acidconjugates, polyethylene glycol, protein or carbohydrate. The associatedor conjugated molecule may also provide bifunctionality to thelipopolysaccharide by, for example, targeting the lipopolysaccharide toa predetermined tissue or cell type, such as T lymphocytes. Theassociation or conjugation between the lipopolysaccharide and the othermolecule may be the result of a direct interaction, such as for example,through a chemical bond or ionic interaction, or alternatively, theassociation or conjugation with the other molecule may be through alinking group. The linking group may be known in the art which serves tolink the lipopolysaccharide, or pharmaceutically acceptable derivativethereof, with the other molecule. Suitable linking groups includesaccharides, oligosaccharides, peptides, proteins, C₂-C₂₀ alkyl,oxyalkylene chains or any other group which does not inhibit the abilityof the lipopolysaccharide of the composition to inhibit HIV replication.The ability of the lipopolysaccharide components of the composition toinhibit HIV replication may be determined by applying assays describedin Section 5.3.

The lipopolysaccharides or pharmaceutically acceptable derivatives ofthe present invention may be monosaccharide precursors of lipid A, suchas lipid X, or may be a tetra-acyl precursor of lipid A, such as lipidIV_(A), etc. Competitive inhibition is typically enhanced by increasedvalence, as once the first contact is made, the probability ofsubsequent contact taking place is favored thermo-dynamically. The useof multivalents is especially useful in blocking low affinity eventswhere high avidity can compensate. Such may be the case for the lipid Aantagonists of the present invention which are thought to function bycompetitively inhibiting LPS from interacting with CD14. In a specificembodiment, the composition comprises multivalent monosaccharideprecursors of lipid A in order to increase the potency and/or biologicalhalf life of the pharmaceutical. In one embodiment, lipid X is found inmultiple copies on a compound for use in the invention.

Multivalent carbohydrates can be prepared using methods known in the artto prepare a branching complex carbohydrate, which conceptuallyresembles a tree in which each branch contains a lipid A precursor, suchas, lipid X. Alternatively, monovalent carbohydrates can be associatedcovalently or noncovalently with a polymer using techniques known in theart (see e.g., Langer et al., International Patent Publication No.WO94/03184, published Feb. 17, 1994, which is herein incorporated byreference in its entirety). The oligosaccharide units of thelipopolysaccharides of the present invention can be bound directly orthrough a linking group to the polymer using known techniques so as toproduce a conjugate in which more than one individual molecule of theoligosaccharide of each lipopolysaccharide is covalently attached.Suitable linking groups include, but are not limited to saccharides,oligosaccharides, peptides, proteins, C₂₋₂₀ alkyl, oxalkylene chains orany other group which does not prevent the lipopolysaccharide frominhibiting HIV replication. Suitable polymers are known in the art andinclude, but are not limited to, a polyol, a polysaccharide, avidin,lipids, lipid emulsions, liposomes, a dendrimer, human serum albumin, aprotein, polylysine, dextran, a glycosaminoglycan, cytclodextrin,agarose, sepharose, and polyacrylamide.

The lipopolysaccharides or pharmaceutically acceptable derivatives ofthe invention may be associated (e.g., ionic interaction) or conjugated(e.g., covalent linkage) with a ligand for a cell-surface molecule so asto target the lipopolysaccharides to tissue or cells expressing thesemolecules. Such oligosaccharide-ligand combinations may be throughdirect interaction of the oligosaccharide and ligand or indirectly usinglinker means known in the art. The oligosaccharide/ligand combinationmay be generated by techniques known in the art (See e.g., Stowell &Lee, 1980, Advances in Carbohydrate Chemistry, 37:225-281) and aregenerated so as not to inhibit the ability of the lipopolysaccharide toinhibit HIV infection. The ability of the lipopolysaccharide/ligandcombination to inhibit HIV replication may routinely be determinedapplying in vitro assays described in Section 5.3 herein and known inthe art. The ability of the lipopolysaccharide/ligand combination tobind to the cell-surface binding partner of the ligand may be determinedusing techniques known in the art. The ligand component of thelipopolysaccharide ligand combination may comprise monoclonal antibody,cell-surface receptor ligand or other homing molecules fortherapeutically significant targets that are known or may routinely beidentified and isolated and/or generated using techniques known in theart. Ligands encompassed by this embodiment include, but are not limitedto, CD4-derived peptide bound by gp120of HIV (from the D1 domain of CD4and distinct from the MHC-binding region (see e.g., Sakihama et al.,1995, PNAS 92:644-648; Ryu et al., 1994, Structure 2:59-74), peptidesderived from the extracellular domain of chemokine receptors (e.g.,CC-CXR-5 or fusion) to which the V3 loop of gp120binds (Choe et al.,1996, Cell 85:1135-1148; Feng et al., 1996, Science 272:872-30 876).

5.2. Synthesis and Isolation of Lipopoltsaccharides

In accordance with the present invention the lipopolysaccharides, LPSanalogs (e.g., antagonists), and lipid A analogs (e.g., antagonists) ofthe present invention can be purified from gram negative microorganismsor produced using classical organic chemistry synthetic techniques knownin the art. Lipid A and LPS antagonists can be identified by theirability to interfere or compete with the activities of LPS or lipid A,e.g., to competitively inhibit the interaction between LPS and thecellular receptor for LPS, for example, as reflected by the inhibitionof a biological consequence of such an interaction. Alternatively, inanother embodiment, the lipopolysaccharide analogs and derivatives ofthe present invention may be prepared by enzymatic processes. Somenon-pyrogenic forms of LPS and lipid A are commercially available (e.g.,from ICN).

5.2.1 Purification of Lipopolysaccharides from Microorganisms

5.2.1.1 Sources of Lipopolysaccharides

Native preparations of non-pyrogenic or reduced-pyrogenic forms of lipidA and LPS amy be obtained from a variety of sources. In accordance withthe present invention, a variety of gram negative strains may be used asthe starting material in producing non-pyrogenic or reduced-pyrogenicforms of LPS and lipid A. Examples of these strains include, but are notlimited to, Haemophilus influenzae, Escherichia coli, Salmonellaenterica, Klebsiella pneumoniae, Bordella pertussis, Pseudomonasaeruginosa, Chlamydia psittaci, Rhodobacter spearoides, it andLegionella pneumophila.

In accordance with the present invention, the non-pyrogenic orreduced-pyrogenic LPS and lipid A preparations may be isolated from gramnegative strains carrying mutations in one of the following genes: kdsA,kdsB, htrB, msbB or both htrB and msbB. The genetics of lipid Abiosynthesis are well described (Raetz, supra; Raetz, Ann. Rev. Biochem59:129-170 (1990); and Schnaitman and Klena, supra). The majority ofmutations that prevent the biosynthesis of lipid A, such as mutations in1pxA, 1pxB, kdsA, kdsB, kdtA, are lethal as the biosynthesis of lipid Ais essential for cell survival (Rick et al., J. Biol. Chem.,252:4904-4912 (1977); Rick and Osborn, J. Biol. Chem., 252:4895-4903(1977); Raetz et al., J. Biol. Chem., 260:16080-16088 (1985); Raetz,supra (1990); Raetz, supra (1993); and Belunis et al., J. Biol. Chem.,270:27646 (1995)). For the most part, therefore, analysis of these geneshas involved the use of temperature-sensitive mutants, which onlydisplay null phenotypes under non-permissive conditions (Rich et al.,supra; Rich and Osborn, supra; Raetz et al., supra; Raetz, supra (1990);Raetz, supra (19930; and Belunis et al., supra). When grown undernon-permissive conditions, 1pxB, kdsA, kdsB, kdsA mutants accumulatenon-pyrogenic precursor forms of LPS (to about 50% of the total LPS),such as lipid X (also called 2,3-diacyl-glucosamine-1-phosphate) orlipid IV_(A) (Raetz et al., supra; Belunis et al., supra).

Alternatively, non-pyrogenic or reduced-pyrogenic LPS and lipid Apreparations may be isolated from gram negative microorganisms carryingat least one mutation in the genes encoding for myristoyl transferase orlauroyl transferase. Gram negative microorganisms carrying mutations inthe pgsA gene, which encodes phosphatidylglycerophospate synthase; 1pxB,the structural gene for disaccharide synthase; the 1pxA gene, encodingUDP-GlcNAc O-acetyltransferase; kdtA the structural gene encoding theKDO (3-deoxy-D-manno-actulosonic acid) transferase, may also be used asa source of non-pyrogenic or reduced-pyrogenic LPS or lipid A structuresin accordance with the present invention.

In a preferred embodiment of the present invention, non-pyrogenic formsof LPS may be isolated from strains of E.coli carrying mutations in bothhtrB and msbB, which produce non-pyrogenic LPS when grown attemperatures above 33° C. and below 44° C. (See PCT InternationalPublication No. WO 97/18837). In a preferred embodiment of the presentinvention, non-pyrogenic LPS is isolated from the E. coli htrB1::Tn10msbB::Ωcam double mutant MLK986. In yet another preferred embodiment,non-pyrogenic preparations of lipid A are isolated from gram negativebacteria KDO-deficient mutants which accumulate lipid IV_(A), anon-pyrogenic form of lipid A (Goldman et al., 1988, J. Bacteriol.170:2185-2192; Raetz et al., 1985, J. Biol. Chem. 260:16080-16088).

Many of the proteins involved in lipid A metabolism are essential tobacteria vitality and therefore mutations in these genes must beintroduced as ones inducible by growth conditions, i.e., temperaturesensitive mutants, or the mutant genes may be under the control of aninducible promoter, such as a tetracycline promoter, tetR, or arepressible promoter, such as a lexA-repressed promoter. Mutations maybe introduced into the genomes of gram negative bacteria using standardrecombinant DNA techniques well known to those of ordinary skill in theart. Mutations in the designated genes listed above may consist of thedeletion of the gene or a portion thereof, insertion of nucleic acidsinto the gene coding region, missense mutations, nonsense mutations(see, e.g., Miller 1992, A Short Course in Bacterial Genetics, ColdSpring Harbor Press; Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, 2d Ed., Cold SpringHarbor, N.Y., Glover), etc. Any technique for mutagenesis known in theart may be used, including but not limited to, chemical mutagenesis, invitro site-directed mutagenesis (Hutchinson, C. et al., 1978, J. Biol.Chem. 253:6551), use of TAB™ linkers (Pharmacia), PCR with mutantprimers, etc.

Further, in vivo cloning techniques can be used to introducemutation-containing nucleic acids into the genomes of gram negativebacteria (see, e.g., Miller 1992, A Short Course in Bacterial Genetics,Cold Spring Harbor Press; Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, 2d Ed., Cold SpringHarbor, N.Y., Glover), etc.

Other bacterial strains may be used in accordance with the presentinvention as a source of forms of LPS and lipid A of reduced or absentpyrogenicity. For example, Rhodobacter sphaeroides LPS has an unusualpentaacyl structure that is significantly less pyrogenic than E. coliLPS in vitro and in vivo (Salimath et al. supra; Qureshi et al. supra,1991; Qureshi et al. supra 1988). R. sphaeroides lipid A (RSLA) is aneffective LPS antagonist (Salimath et al. supra) and prevents LPSinduced lethality in mice. Bacterial strain Rhizobium leguminosarum mayalso be used as a source of non-pyrogenic form of LPS and lipid A, inaccordance with the present invention. Lipid A isolated from R.leguminosarum lacks phosphate altogether and does not contain aglucosamine disaccharide (Bhat et al. 1992 Glycobiology 2:535-539) andis a very potent antagonist of lipid A. R. leguminosarum lipid Acontains a 2-deoxy-2-aminogluconate residue in place of the glucosamine1-phosphate at the reducing end, and bears a glacturonic acid moietyinstead of a monophosphate at position 4′.

5.2.1.2 Isolation of Lipopolysaccharides

The lipopolysaccharides of the present invention may be purified fromgram negative microorganisms using any technique known in the art. Inthis embodiment, the lipopolysaccharides of the present invention areisolated from the cell wall of the bacteria. In gram-negative bacteria,the cell wall is far more complex than for gram positives and containsglycopeptide, lipopolysaccharide, phospholipid and protein. Up to 20% ofthe wall content may be lipids but only a proportion of these arereadily extractable by conventional solvent methods. That is due to thecovalent nature of the lipopolysaccharide linkages. In spite of theforegoing, in accordance with the present invention, thelipopolysaccharides of the present invention may be isolated fromgram-negative bacteria using the procedures described infra.

By way of example, and not by limitation, the lipopolysaccharides of thepresent invention may be isolated by the following method:

Bacterial strains are cultured on solid media both at 30° C., 37° C. or42° C. prior to seeding the liquid media. Liquid cultures (1L) areseeded the following day at a starting inoculum of ca. 1×10⁴ cfu/ml andgrown for 16 hr at 30° C., 37° C. or 42° C. with shaking (250 opm).

The liquid cultures are harvested by centrifugation at 7000×g for 10min, washed once in 250 ml endotoxin-free irrigation saline (Baxter) andthe weight of the bacterial pellets was determined. The pellets then areresuspended in endotoxin-free water (Baxter) at a final density of 2%w/v±0.25%. Subsequently, LPS is isolated by two cycles of hot-waterphenol extraction. In short, the bacterial suspensions are heated to 70°C. to which an equal volume of pre-warmed phenol is added and mixed for15 min at 70° C. The mixtures are cooled to 25° C. and then centrifugedat 18,000×g for 15 min. Following this centrifugation the aqueous phasesare removed, placed into dialysis tubing (SpectraPor) and dialyzedagainst running distilled H₂O overnight. The retentates are then placedinto fresh 50 ml polypropylene tubes and treated with RNaseA (100 μg/ml)at 37° C. for 1 hr, followed by DNaseI (50 μg/ml and 5 mM Mgcl₂) at 37°C. for 1 hr, followed by Pronase (250 μg/ml) at 37° C. for 1 hr. ThenEDTA is added to a final concentration of 5 mM and the hot-water phenolextraction procedure described above is repeated. Following dialysis theretentates are centrifuged at 20,000×g for 15 min at 4° C. Thesupernatants are transferred to fresh Beckman 50Ti tubes and the LPS ispelleted by centrifugation at 110,000×g for 2H at 4° C. The supernatantsare discarded and the pellets are vacuum dried. Each LPS preparation isevaluated for DNA and protein contamination by standard techniques inthe art, such as SDS-PAGE and silver stain, BCA protein estimate assayand UV spectrophotometry.

Also included within the scope of the present invention are LPS andlipid A molecules which are differentially modified during or aftersynthesis to yield reduced or absent pyrogenic properties of thepreparation. In specific embodiments, the LPS and lipid A molecules aretreated by alkaline hydrolysis or acyloxyacyl hydrolase. Any of numerouschemical modifications may be carried out by known techniques, such asacylation, deacylation, formylation, oxidation, reduction, etc.

5.2.2 Synthesis of Lipopolysaccharides

Potent synthetic lipid A molecules with strong LPS antagonist propertiesand of reduced or absent pyrogenicity may be synthesized by a variety oforganic chemistry synthetic techniques. In one embodiment of the presentinvention, the synthetic lipid A and LPS molecules are modeled after LPSmolecules of reduced or absent pyrogenicity which occur in nature andmolecules with strong LPS antagonist activities which occur in nature.

The lipopolysaccharide is a complex polymer in four parts. Outermost isa carbohydrate chain variable length (called the O-antigen) which isattached to a core polysaccharide. The core polysaccharide is dividedinto the outer core and the backbone. These two structures vary betweenbacteria. Finally the backbone is attached to a glycolipid called lipidA. The link between lipid A and the rest of the molecule is usually viaa number of 3-deoxy-D-manno-octulosonic acid (KDO) molecules. Thepresence of KDO is often used as a marker for lipopolysaccharide (orouter membrane) even though it is not present in all bacteriallipopolysaccharides. The phosphate and 3-deoxy-D-mannooctulosomic acid(KDO) molecules (the presence of KDO is often used as a marker forlipopolysaccharide) are also substituted. Unsaturated and cyclopropanefatty acids which are common in other lipid types are absent fromlipopolysaccharide.

Lipid A is composed of a disaccharide of glucosamines. The amino groupsare substituted with 3-hydroxymristate while hydroxyl groups containsaturated (12-16 carbon) acids and 3-myristoxymyristate.Lipopolysaccharides and lipid A may be obtained from commercial sources,e.g., from Sigma. However, by way of example, but not by way oflimitation, lipolysaccharides may be synthesized as follows: hydroxyacids and disaccharides are condensed followed by addition of saturatedfatty acids. The hydroxy fatty acids may come from acetyl CoA whereasCMP-KDO may serve as the source of the second additional units. Afterthe addition of saturated fatty acids, sugars are added from nucleotidediphosphate derivatives.

The O-antigen is may be synthesized in three stages. For example, butnot by way of limitation, the oligosaccharide units are transferred fromnucleotide diphosphate carriers to a galactose attached to another lipidcarrier. The oligosaccharide units are then polymerized and lipidcarriers are released in the process. Finally the complete O-antigen istransferred to the R-core with the release of an isoprenoid carrier.

For an overview of the synthesis of lipopolysaccharides and lipid Astructures, see, e.g., Raetz, 1993, J. Bacteriology 175:5745-5753. (Seealso U.S. Pat. Nos. 5,593,969 and 5,191,072).

The polysaccharide unit may also be synthesized with donor saccharidemoieties and acceptor moieties which are commercially available and/ormay be synthesized through organic synthesis applying techniques knownin the art. Activated saccharides generally consist of uridine orguanosine diphosphate and cytidine monophosphate derivatives of thesaccharides in which the nucleoside mono and diphosphate serves as aleaving group. Thus, the activated saccharide may be a saccharide-UDP, asaccharide-GDP, or a saccharide-CMP. Nucleoside monophosphates arecommercially available, may be prepared from known sources such asdigested yeast RNA (see e.g., Leucks et al,. 1979, J. Am. Soc.101:5829), or routinely prepared using known chemical synthetictechniques (see e.g., Heidlas et al., 1992, Acc, Chem. Res. 25:307;Kochetkov et al., 1973, Adv. Carbohydr. Chem. Biochem. 28:307). Thesenucleoside monophosphates may then be routinely transformed intonucleoside diphosphates by kinase treatment. For review, see Wong etal., 1994, Enzymes in Synthetic Organic Chemistry, Pergamon Press,Volume 12, pp. 256-264.

Glycosyltransferase enzymes for synthesizing the compositions of theinvention can be obtained commercially or may be derived from biologicalfluids, tissue or cell cultures. Such biological sources include, butare not limited to, pig serum and bovine milk. Glycosyltransferases thatcatalyze specific glycosidic linkages may routinely be isolated andprepared as described in International Patent Publication No. WO93/13198 (published Jul. 8, 1993). Alternatively, theglycosyltransferases can be produced through recombinant or synthetictechniques known in the art (For review, see Wong et al., 1994, Enzymesin Synthetic Organic Chemistry, Pergamon Press, Volume 12, pp. 275-279).

The compositions of the invention are preferably synthesized usingenzymatic processes (see e.g., U.S. Pat. No. 5,189,674, andInternational Patent Publication No. 91/16449, published Oct. 31, 1991).Briefly, a glycosyltransferase is contacted with an appropriateactivated saccharide and an appropriate acceptor molecule underconditions effective to transfer and covalently bond the saccharide tothe acceptor molecule. Conditions of time, temperature, and pHappropriate and optimal for a particular saccharide unit transfer can bedetermined through routine testing; generally, physiological conditionswill be acceptable. Certain co-reagents may also be desirable; forexample, it may be more effective to contact the glycosyltransferasewith the activated sugar and the acceptor molecule in the presence of adivalent cation. Optionally, an apparatus as described by U.S. Pat. No.5,288,637, is used to prepare such compositions.

While glycosyltransferases are highly stereospecific andsubstrate-specific, minor chemical modifications are tolerated on boththe donor and acceptor components. Accordingly, the oligosaccharidecomponents of the invention may be synthesized using acceptor and/ordonor components that have been modified so as not to interfere withenzymatic formation of the desired glycosidic linkage. The ability ofsuch a modification not to interfere with the desired lycosidic linkagemay routinely be determined using techniques and bioassays known in theart, such as, for example, labelling the carbohydrate moiety of theactivated sugar donor, contacting the acceptor and donor moieties withthe glycosyltransferase specific for forming the glycosidic linkagebetween the donor and acceptor moieties, and determining whether thelabel is incorporated into the molecule containing the acceptor moiety.

Also included within the scope of the present invention are LPS andlipid A molecules which are differentially modified during or aftersynthesis to enhance or yield of reduced or absent pyrogenicity of thepreparation. In specific embodiments, the LPS and lipid A molecules aretreated by alkaline hydrolysis or acyloxyacyl hydrolase. Any of numerouschemical modifications may be carried out by known techniques, such asacylation, deacylation, formylation, oxidation, reduction, etc.

It is also within the scope of this invention, to synthesize analogs oflipid a having one or more acyloxyacyl groups removed. Lipid A, eitherchemically synthesized or isolated from a gram negative microorganismmay be treated with acyloxyacyl hydrolase in order to achieve or enhancethe non-pyrogenic properties of the preparation. Acyloxyacyl hydrolasehydrolyzes the ester bonds between non-hydroxylated fatty acids and the3-hydroxy functions of 3-hydroxy fatty acids bound in ester or amidelinkages to glucosamine disaccharide of lipid A.

It is further within the scope of this invention, to synthesize analogsof lipid A and LPS having one or more non-hydroxylated fatty acidsremoved. Lipid A or LPS either chemically synthesized or isolated from agram negative microorganism may be deacylated in order to achieve orenhance the substantially reduced or absent pyrogenicity of thepreparation.

5.3 Therapeutic Uses

The invention provides for treatment or prevention of diseases anddisorders associated with immunodeficiency virus infection, including,but not limited to, human immunodeficiency virus (HIV), simianimmunodeficiency virus (SIV), and feline immunodeficiency virus (FIV),by administration of a therapeutic compound (termed herein“Therapeutic”). Such “Therapeutics” include, but are not limited to:preparations of LPS or lipid A of reduced or absent pyrogenicity, andanalogs and derivatives and antagonists thereof, of reduced or absentpyrogenicity. By way of example, but not limitation, the antagonists canbe derived from gram negative bacteria, or gram-negative bacteriacontaining at least one mutation selected from the group consisting ofkdsA, kdsB, htrB, msbb and derivative and analogs of the foregoing; LPSorlipid A preparations which have been modified to enhance or yieldreduced or absent pyrogenicity, including, but not limited tomonophosphoryl lipid A, penta-acyl lipid A, lipid X, lipid IV_(A) orlipid A or LPS derived from deacylation, treatment with acyloxyaclhydroxylase, or by treatment with an alkaline, and derivatives andanalogs of the foregoing; synthetic lipid A and LPS antagonists, such aslipid X, lipid IV_(A) and prophylactically and therapeutically effectiveLPS and lipid A analogs and derivatives thereof.

It is also within the scope of this invention, to use therapeutically orprophylactically monosaccharide analogs of lipid A and LPS. Lipid A orLPS structures to be used therapeutically or prophylactically inaccordance with the present invention can be either chemicallysynthesized or isolated from a gram negative microorganism, in which theester bond of LPS and lipid A is catalyzed or the glycosidic bond of LPSand lipid A is catalyzed resulting in a monosaccharide derivative withenhanced non-pyrogenic properties.

It is also within the scope of this invention, to use mixtures of lipidA or LPS structures and antagonists and analogs and derivatives thereoftherapeutically or prophylactically in accordance with the presentinvention.

In another embodiment of the present invention, a mixture of one lipid Aor LPS structure of the present invention mixed with at least one otherLPS or lipid A structure of the present invention can be used to treator prevent immunodeficiency virus infection, including HIV infectionsand disorders associated therewith. In accordance with the presentinvention, a mixture comprising at least one LPS antagonist and an LPSor lipid A structure isolated from a gram negative bacteria, wherein theLPS antagonist is in molar excess of the LPS or lipid A structure can beused to treat or prevent HIV infection and disorders associated therein.For example, an LPS or lipid A antagonist combined with a fully activeLPS or lipid A, or a monophosphate LPS or lipid A, or a second LPS orlipid A antagonist.

Examples of Therapeutics are those lipopolysaccharides described inSection 5.1 and 5.2.

A preferred embodiment of the invention relates to methods of using aTherapeutic for treatment or prevention of HIV infection, preferablyHIV-1 infection, in a human subject. In a specific embodiment, theTherapeutic is used for the treatment or prevention of HIV infection ina human subject who suffers from Kaposi's sarcoma (KS). In the treatmentof HIV infection, the Therapeutic of the invention can be used toprevent progression of HIV-1 infection in a seropositive patient to ARCor to AIDS in the patient, or to treat a human patient with ARC or AIDS.

In a preferred aspect of the invention, preparations of reduced orabsent pyrogenicity of LPS and/or lipid A and/or derivatives and/oranalogs thereof are used to treat HIV infection. The utility of suchpreparations may be determined by the in vitro and in vivo assaysdescribed in Section 5.5 infra or by any other method known in the art.

5.4 Combination Therapy

According to a specific embodiment of the present invention, apreparation of reduced or absent pyrogenicity of LPS or lipid A or ananalog or derivative thereof, an inhibitor of HIV viral replication, mayoptionally be used in combination with other therapeutic agents toenhance the antiviral effect achieved. Preferably a non-pyrogenicpreparation of LPS or lipid A or an analog or derivative thereof is usedin combination with another antiviral agent. Such additional antiviralagents which may be used with a preparation of reduced or absentpyrogenicity of LPS or lipid A or analog or derivative thereof includebut are not limited to those which function on a different targetmolecule involved in viral replication, e.g., reverse transcriptaseinhibitors, viral protease inhibitors, glycosylation inhibitors; thosewhich act on a different target molecule involved in viral transmission;those which act on a different loci of the same molecule; and thosewhich prevent or reduce the occurrence of viral resistance. One skilledin the art would know of a wide variety of antiviral therapies whichexhibit the above modes of activity.

A preparation of reduced or absent pyrogenicity of LPS or lipid A or ananalog or derivative thereof can also be used in combination withretrovirus inhibitors, such as nucleoside derivatives. Nucleosidederivatives are modified forms of purine and pyrimidine nucleosideswhich are the building blocks of RNA and DNA. Many of the nucleosidederivatives under study as potential anti-HIV medications result inpremature termination of viral DNA replication before the entire genomehas been transcribed. These derivatives lack 3′ substituents that canbind to subsequent nucleosides and result in chain termination.Nucleoside derivatives such as 3′ azido-3′-thymidine (AZT) anddideoxyinosine (ddI) have been exploited as inhibitors of HIV-1replication, both in vitro and in vivo. Nucleoside analogs are currentlythe only licensed therapeutics for the treatment of HIV infection andAIDS (Fischl et al, 1987 N. Engl. J. Med. 317, 185-191; Mitsuya andBroder, 1987 Nature 325, 773-778). This class of compounds works byinhibiting reverse transcriptase resulting in a block in cDNA synthesis(Mitsuya and Broder, 1987), these inhibitors work early in theinfectious cycle of HIV-1 and inhibit integration into T-cell genome.However, AZT therapy leads to development of resistant HIV strains(Larder 1989, 1991, Ibid.) and demonstrates toxicity in AIDS patientsupon long-term therapy (Fischl et al., 1987, N. Engl. J. Med.317:185-191; Creagh-Kirk, et al., 1988, J.A.M.A. 260:3045-3048).

Further, a preparation of reduced or absent pyrogenicity of LPS or lipidA or a derivative or analog thereof can be used in combination withnucleoside derivatives which include but are not limited to,2′,3′-dideoxyadenosine (ddA); 2′,3′-dideoxyguanosine (ddG);2′,3′-dideoxyinosine (ddI); 2′,3′-dideoxycytidine (ddC);2′,3′-dideoxythymidine (ddT); 2′,3′-dideoxy-dideoxythymidine (d4T) and3′-azido-2′,3′-dideoxythymidine (AZT). Alternatively, halogenatednucleoside derivatives may be used, preferably2′,3′-dideoxy-2′-fluoronucleosides including, but not limited to,2′,3′-dideoxy-2′-fluoroadenosine; 2′,3′-dideoxy-2′-fluoroinosine;2′,3′-dideoxy-2′-fluorothymidine; 2′,3′-dideoxy-2′-fluorocytosine; and2′,3′-dideoxy-2′,3′-didehydro-2′-fluoronucleosides including, but notlimited to 2′,3′-dideoxy-2′,3′-didehydro-2′-fluorothymidine (Fd4T).Preferably, the 2′,3′-dideoxy-2′-fluoronucleosides of the invention arethose in which the fluorine linkage is in the beta configuration,including, but not limited to, 2′3′-dideoxy-2′-beta-fluoroadenosine(F-ddA), 2′,3′-dideoxy-2′-beta-fluoroinosine (F-ddI), and2′,3′-dideoxy-2′-beta-fluorocytosine (F-ddC). Such combinations allowone to use a lower dose of the nucleoside derivative thus reducing thetoxicity associated with that agent, without loss of antiviral activitybecause of the use of the non-pyrogenic preparation of LPS or lipid A.Moreover, such a combination reduces or avoids viral resistance.

Preferred combinations of preparations of reduced or absent pyrogenicityof LPS or lipid A or derivatives or analogs thereof and nucleosidederivatives within the scope of the present invention include aneffective amount of a preparation of reduced or absent pyrogenicity ofLPS or lipid A or analogs or derivatives thereof and an effective amountof AZT to treat HIV infection; and an effective amount of a preparationof reduced or absent pyrogenicity of LPS or lipid A or derivative andanalogs thereof and an effective amount of ddI.

According to the present invention, preparations of reduced or absentpyrogenicity of LPS or lipid A or derivatives and analogs thereof canalso be used in combination with uridine phosphorylase inhibitors,including but not limited to acyclouridine compounds, includingbenzylacyclouridine (BAU); benzyloxybenzylacyclouridine (BBAU);aminomethyl-benzylacyclouridine (AMBAU);aminomethyl-benzyloxybenzylacyclouridine (AMB-BAU);hydroxymethyl-benzylacyclouridine (HMBAU); andhydroxymethyl-benzyloxybenzylacyclouridine (HMBBAU).

According to the present invention, preparations of; reduced or absentpyrogenicity of LPS or lipid A or derivatives and analogs thereof can beused in combination with viral protease inhibitors, including but notlimited to, Invirase (saquinavir, Roche), ABT-538 (Abbott, CAS Reg. No.155213-67-5), AG1343 (Burroughs Wellcome/Glaxo, CAS Reg. No.161814-49-9). Protease inhibitors are generally thought to workprimarily during or after assembly (i.e., viral budding) to inhibitmaturation of virions to a mature infectious state. For example, ABT-538has been shown to have potent antiviral activity in vitro and favorablepharmokinetic and safety profiles in vivo (Ho, et al., 1995, Nature373:123-126). Administration of ABT-538 to AIDS patients causes plasmaHIV-1 levels to decrease exponentially and CD4 lymphocyte counts to risesubstantially. The exponential decline in plasma viraemia followingABT-538 treatment reflects both the clearance of free virions and theloss of HIV-1 producing cells as the drug substantially blocks newrounds of infection. ABT-538 treatment reduces virus-mediateddestruction of CD4 lymphocytes. Combining this treatment with apreparation of reduced or absent pyrogenicity of LPS or lipid A or ananalog or derivative thereof, which inhibits at an earlier stage of HIVinfection, viral fusion, would be likely to have synergistic effects andhave a dramatic clinical impact.

In order to evaluate potential therapeutic efficacy of reduced-pyrogenicor non-pyrogenic preparations of LPS or lipid A or derivatives andanalogs thereof in combination with the antiviral therapeutics describedabove, these combinations may be tested for antiviral activity accordingto methods known in the art.

A compound of the invention can be administered to a human patient byitself or in pharmaceutical compositions where it is mixed with suitablecarriers or excipients at doses to treat or ameliorate variousconditions involving HIV-infection. A therapeutically effective dosefurther refers to that amount of the compound sufficient to inhibit HIVinfection. Therapeutically effective doses may be administered alone oras adjunctive therapy in combination with other treatments for HIVinfection or associated diseases. Techniques for the formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton,Pa., latest addition.

In another embodiment, HIV infection is treated or prevented byadministration of a Therapeutic of the invention in combination with oneor more chemokines. In particular, the Therapeutic is administered withone or more C—C type chemokines, especially one or more from the groupRANTES, MIP-1α and MIP-1β).

5.5 Demonstration of Therapeutic Utility

The present invention relates to assaying the therapeutic compounds ofthe present invention for their therapeutic effectiveness, includingassaying their pyrogenicity properties in addition to their ant-HIVproperties

Such assays include, but are not limited to:

5.4.1 Determining the Pyrogenicity of the Preparation

The Therapeutics of the invention are preferably tested in vitro, andthen in vivo for non-pyrogenicity or reduced pyrogenicity, prior to usein humans. Any in vitro or in vivo assay known in the art to measurepyrogenic, inflammatory or endotoxic activity can be used to test thepyrogenicity of a Therapeutic of the invention. By way of example, andnot by way of limitation, one could use any of the in vitro assaysdescribed infra in Section 6.

In an embodiment of the invention, a method of screening a preparationcomprising LPS or lipid A or a derivative or analog thereof forpyrogenic properties comprises assaying said preparation for the abilityto induce secretion of pyrogenic cytokines. In one specific embodiment,the preparation comprising LPS or lipid A or a derivative or analogthereof is assayed by a method comprising measuring cytokine levelssecreted from peripheral blood monocytes, which cells have beencontacted with the preparation and comparing to levels of cytokinessecreted from cells not contacted with the preparation and/or levels ofcytokines secreted from cell contacted with E. coli LPS (known to behighly pyrogenic). Quantitation of TNFα, IL-1β or IL-6 in culturesupernatants can be achieved by capture ELISA. In another specificembodiment, the preparation comprising LPS or lipid A or a derivative oranalogue thereof is assayed by a method comprising measuring β chemokinelevels secreted from peripheral blood monocytes, which cells have beencontacted with the preparation and comparing to levels of β chemokinessecreted from cells not contacted with the preparation and/or levels ofchemokines secreted from cells contacted with E. coli LPS. Quantitationof MIP-1α, MIP-1β and RANTES in culture supernatants can be achieved bycapture ELISA.

The assays described above may be carried out by any method known in theart. By example, and not by limitation, the assays described above maybe carried out as follows:

-   -   50 ml of whole blood is mixed with an equal volume of RPMI (Life        Technologies) and PBMCs are isolated by density gradient using        lymphocyte separation medium according to the manufacturer's        directions (Organon). The PBMCs are washed twice with RPMI then        resuspended in 6 ml of ice cold sterile water and placed on ice        for 30 sec to lyse the erythrocytes. The osmolarity is adjusted        by adding 2 ml of ice cold 3.5% (w/v) NaCl; the PBMCs are        harvested by centrifugation, washed with RPMI and resuspended in        complete medium (CM; RPMI containing pyruvate, glutamine,        PenStrep, and 10% endotoxin-free human AB serum (Life        Technologies) at a density of 6×10⁶ PBMCs/ml. CM containing the        preparations to be assayed are placed into duplicate wells of a        96-well flat bottom culture late (Costar) at double the target        final concentration. An equal volume of CM containing the PBMCs        then is added to these wells and the culture plates are        incubated at 37° C. in 5% CO₂ for 8 hr. The supernatants are        then removed and stored at 70° C.

Quantitation of TNFα, IL-1β, IL-6, MIP-1α, MIP-1β and RANTES in theseculture supernatants is achieved by capture ELISA (R&D Systems).

The Therapeutics of the invention may also be tested in vivo fornon-pyrogenicity prior to use in humans. For example, pyrogenic activitymay be measured in vivo by a dermal Schwartzman reaction and the rabbitpyrogen test. By way of example but not limitation, this is performed asfollows: New Zealand rabbits may receive an intradermal injection of thepreparation of LPS or lipid A or a derivative or analog thereof(approximately 2.5 μg), followed by an intravenous dose (2-4 μg/kg) 24hours later. The dermal lesions are scored 4 to 6 hours later andcompared to rabbits which received an infection of a preparation of LPSfrom E. coli. The therapeutics of the invention may also be determinedby the rabbit pyrogen test. The thermal response index (TRI) for LPSpreparations is determined by injecting a New Zealand white rabbitweighing 3-4 kg with an intravenous dose of approximately 50 ng LPS.Temperature is monitored with a rectal probe and recorded every 10minutes. The TRI is the integrated product of the temperature abovebaseline (0C) and time (degree-hours) (Zimmer et al., 1981 Peptides2:413).

5.4.2 Determining the ANTI-HIV Activity of the Preparation

The Therapeutics of the invention are preferably tested in vitro, andthen in vivo for the desired therapeutic or prophylactic activity, priorto use in humans. Any in vitro or in vivo assay known in the art tomeasure HIV infection or production can be used to test the efficacy ofa Therapeutic of the invention. By way of example, and not by way oflimitation, one could use any of the in vitro or in vivo assaysdescribed infra in Section 6.

In an embodiment of the invention, a method of screening a preparationcomprising a lipopolysaccharide, i.e. lipid A or LPS or lipid Aantagonist or LPS antagonist, having non-pyrogenic properties or anyderivative or analogue thereof, for anti-HIV activity is provided, whichassay comprises assaying said preparation for the ability to inhibit HIVreplication or expression of HIV RNA or protein. In one specificembodiment, the lipopolysaccharide preparation is assayed by a methodcomprising measuring HIV-1 p24 antigen levels in cultured hematopoieticcells which have been contacted with the lipopolysaccharide preparationprior to infection with HIV-1, and comparing the measured HIV-1 p24antigen levels in the cells which have been contacted with thelipopolysaccharide preparation with said levels in cells not socontacted with the preparation, wherein a lower level in said contactedcells indicates that the preparation has anti-HIV activity. In anotherspecific embodiment, the lipopolysaccharide preparation is assayed by amethod comprising measuring the activity of a reporter gene productexpressed from a construct in which the HIV-1 LTR is operably linked tosaid reporter gene, wherein said construct is present in cells whichhave been contacted with the preparation; and comparing the measuredexpression of said reporter gene in the cells which have been contactedwith the preparation with said levels in such cells not so contacted,wherein a lower level in said contacted cells indicates that thepreparation has anti-HIV activity. In another specific embodiment, thelipopolysaccharide preparation is assayed by a method comprisingmeasuring HIV-1 derived RNA transcripts or HIV-1 antigen levels in HIV-1transgenic mice administered the preparation; and comparing the measuredtranscript or antigen levels in the mice which have been administeredthe preparation with said levels in mice not so administered, wherein alower level in said administered mice indicates that the preparation hasanti-HIV activity. In yet another specific embodiment, thelipopolysaccharide preparation is assayed by a method comprisingmeasuring SIV p27 antigen levels in the peripheral blood mononuclearcells of SIV infected monkeys administered the preparation; andcomparing the measured antigen levels in the monkeys which have beenexposed to the preparation with said levels in monkeys not soadministered, wherein a lower level in said administered monkeysindicates that the preparation has anti-HIV activity.

By way of example, to assay a Therapeutic in vitro, one can examine theeffect of the Therapeutic on HIV replication in cultured cells. Briefly,cultured hematopoietic cells (e.g., primary PBMCs, isolated macrophages,isolated CD4⁺ T cells or cultured H9 human T cells) are acutely infectedwith HIV-1 using titers known in the art to acutely infect cells invitro, such as 10⁵ TCID₅₀/ml. Then, appropriate amounts of theTherapeutic are added to the cell culture media. Cultures are assayed 3and 10 days after infection for HIV-1 production by measuring levels ofp24 antigen using a commercially available ELISA assay. Reduction in p24antigen levels over levels observed in untreated controls indicates theTherapeutic is effective for treatment of HIV infection.

Additionally, assays for HIV-1 LTR driven transcription are useful fortesting the efficacy of Therapeutics of the invention. Specifically, areporter gene, i.e., a gene the protein or RNA product of which isreadily detected, such as, but not limited to, the gene forchloramphenicol acetyltransferase (CAT), is cloned into a DNA plasmidconstruct such that the transcription of the reporter gene is driven bythe HIV-1 LTR promoter. The resulting construct is then introduced bytransfection, or any other method known in the art, into a cultured cellline, such as, but not limited to, the human CD4⁺ T cell line HUT78.After exposure of the transformed cells to the Therapeutic,transcription from the HIV-1 LTR is determined by measurement of CATactivity using techniques which are routine in the art. Reduction inHIV-1 LTR driven transcription demonstrates utility of the Therapeuticfor treatment and/or prevention of HIV infection.

Exemplary tests in animal models are described briefly as follows:First, a Therapeutic of the invention is administered to mice transgenicfor HIV-1, e.g., mice which have integrated molecular clone pNL4-3containing 7.4 kb of the HIV-1 proviral genome deleted in the gag andpol genes (Dickie, P., et al., 1991, Virology 185:109-119). Skinbiopsies taken from the mice are tested for HIV-1 gene expression byRT-PCR (reverse transcription-polymerase chain reaction) or for HIV-1antigen expression, such as expression of gp120or NEF, byimmunostaining. Additionally, the mice are examined for reduction in thecachexia and growth retardation usually observed in HIV-1 transgenicmice (Franks, R. R., et al., 1995, Pediatric Res. 37:56-63).

The efficacy of Therapeutics of the invention can also be determined inSIV infected rhesus monkeys (see Letrin, N. L., and King, N. W., 1990,J. AIDS 3:1023-1040), particularly rhesus monkeys infected withSIV_(mac251), which SIV strain induces a syndrome in experimentallyinfected monkeys which is very similar to human AIDS (Kestler, H., etal., 1990, Science 248:1109-1112). Specifically, monkeys can be infectedwith cell free SIV_(mac251), for example, with virus at a titer of10^(4.5) TCID₅₀/ml. Infection is monitored by the appearance of SIV p27antigen in PBMCs. Utility of the Therapeutic is characterized by normalweight gain, decrease in SIV titer in PBMCs and an increase in CD4⁺ Tcells.

Once the Therapeutic has been tested in vitro, and also preferably in anon-human animal model, the utility of the Therapeutic can be assayed inhuman subjects. The efficacy of treatment with a Therapeutic can beassessed by measurement of various parameters of HIV infection and HIVassociated disease. Specifically, the change in viral load can bedetermined by quantitative assays for plasma HIV-1 RNA usingquantitative RT-PCR (Van Gemen, B., et al., 1994, J. Virol. Methods49:157-168; Chen, Y. H., et al., 1992, AIDS 6:533-539) or by assays forviral production from isolated PBMCs. Viral production from PBMCs isdetermined by co-culturing PBMCs from the subject with H9 cells andsubsequent measurement of HIV-1 titers using an ELISA assay for p24antigen levels (Popovic, M., et al., 1984, Science 204:309-25 321).Another indicator of plasma HIV-1 levels and AIDS progression is theproduction of inflammatory cytokines such as IL-6, IL-8 and TNF-α; thus,efficacy of the Therapeutic can be assessed by ELISA tests for reductionof serum levels of any or all of these cytokines. Administration of theTherapeutic can also be evaluated by assessing changes in CD4⁺ T celllevels, body weight, or any other physical condition associated with HIVinfection or AIDS or AIDS Related Complex (ARC). Reduction in HIV viralload or production, increase in CD4⁺ T cell or amelioration ofHIV-associated symptoms demonstrates utility of a Therapeutic foradministration in treatment/prevention of HIV infection.

5.6 Therapeutic Compositions and Methods of Administration

The invention provides methods of treatment and prevention byadministration to a subject in need of such treatment of atherapeutically or prophylactically effective amount of a Therapeutic ofthe invention. The subject is preferably an animal, including, but notlimited to, animals such as primates, monkeys, cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human. In a specific embodiment, the subject is a humanafflicted with HIV infection or related disorders. In a preferredembodiment, the Therapeutic is purified (i.e., separated from componentswith which it is associated in its natural environment).

Various delivery systems are known and can be used to administer aTherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeuticnucleic acid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved, for example and not by way oflimitation, by topical application, by injection, by means of acatheter, by means of a suppository, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers.

In another embodiment, the Therapeutic can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) In yet anotherembodiment, the Therapeutic can be delivered in a controlled releasesystem. In one embodiment, a pump may be used (see Langer, supra;Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Florida (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier. As used herein,the term “pharmaceutically acceptable carrier” refers to a carriermedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient, is chemically inert and is not toxicto the patient to whom it is administered. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the Therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the Therapeutic, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The Therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vivo and/or in vitroassays may optionally be employed to help predict optimal dosage ranges.The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the disease ordisorder, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Predicted suitable dosesof a non-pyrogenic preparation of LPS or lipid A or derivatives oranalogs thereof for treatment or prevention of HIV infection include,but are not limited to, 1 ng/kg to 2 mg/kg per week. Routes ofadministration of a Therapeutic include, but are not limited to,intramuscularly, subcutaneously or intravenously. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

6. EXAMPLE NON-PYROGENIC LPS STIMULATES β CHEMOKINE SECRETION IN PBMC

These experiments were carried out to demonstrate the ability ofnon-pyrogenic LPS antagonists to competitively inhibit the interactionbetween LPS and the cellular receptor for LPS. Recent biochemicalevidence suggests that the htrB and msbb genes serve as lauroyl andmyristoyl transferases, respectively (Rietschel et al., 1994 FASEB J.8:217-225; Summerville et al., 1996, J. Clin. Invest. 97: 359-365; Leeet al., 1995, J. Biol. Chem. 270:27151-27159). In support, massspectrometry analysis of LPS from an E. coli msbB mutant showed thatthis mutant produced a pentaacyl lipid A that lacked the ester-linkedmyristoyl side chain (Lee et al., supra). Similarly, an htrB mutant ofHaemophilus influenzae produced a mixture of pentaacyl and tetraacyllipid A. Thus, HtrB and MsbB transferase activities are believed to benecessary for the generation of LPS from KDO₂-lipid IV_(A) (Summervilleet al., supra). These experiments tested whether an E. coli htrB msbBdouble mutant would accumulate LPS that contained lipid A partialstructures and that would serve as a convenient source of LPSantagonist.

6.1 Materials and Methods

Cell Culture

Strains W3110 and MLK986 were cultured on solid media both at 30° C.,37° C. or 42° C. prior to seeding the liquid media. Liquid cultures (1L)were seeded the following day at a starting inoculum of ca. 1×10⁴ cfu/mland grown for 16 hr at 30° C., 37° C. or 42° C. with shaking (250 opm).

Isolation of LPS

The liquid cultures were harvested by centrifugation at 7000×g for 10min, washed once in 250 ml endotoxin-free irrigation saline (Baxter) andthe weight of the bacterial pellets was determined. The pellets thenwere resuspended in endotoxin-free water (Baxter) at a final density of2% w/v±0.25%. Subsequently, LPS was isolated by two cycles of hot-waterphenol extraction. In short, the bacterial suspensions were heated to70° C. to which an equal volume of pre-warmed phenol was added and mixedfor 15 min at 70° C. The mixtures were cooled to 25° C. and thencentrifuged at 18,000×g for 15 min. Following this centrifugation theaqueous phases were removed, placed into dialysis tubing (SpectraPor)and dialyzed against running distilled H₂O overnight. The retentateswere then placed into fresh 50 ml polypropylene tubes and treated withRNaseA (100 μg/ml) at 37° C. for 1 hr, followed by DNaseI (50 μg/ml and5 mM Mgcl₂) at 37° C. for 1 hr, followed by Pronase (250 μg/ml) at 37°C. for 1 hr. Then EDTA was added to a final concentration of 5 mM andthe hot-water phenol extraction procedure described above was repeated.Following dialysis the retentates were centrifuged at 20,000×g for 15min at 4° C. The supernatants were transferred to fresh Beckman 50Titubes and the LPS was pelleted by centrifugation at 110,000×g for 2H at4° C. The supernatants were discarded and the pellets were vacuum dried.Each LPS preparation was evaluated for DNA and protein contamination bySDS-PAGE and silver stain, BCA protein estimate assay and UVspectrophotometry.

PBMC Activation Assays

PBMCs were obtained from 50 ml of whole blood as described above andresuspended in complete medium (CM; RPMI containing pyruvate, glutamine,PenStrep, and 10% endotoxin-free human AB serum (Life Technologies) at adensity of 6×10⁶ PBMCs/ml. CM containing W3110 LPS, MLK986 LPS orsynthetic lipid IV_(A) was placed into duplicate wells of a 96-well flatbottom culture late (Costar) at double the target final concentration.An equal volume of CM containing the PBMCs then was added to these wellsand the culture plates were incubated at 37° C. in 5% CO₂ for 8 hr. Thesupernatants were then removed and stored at 70° C. Quantitation ofTNFα, IL-1β, IL-6, MIP-1α and MIP-1β in these culture supernatants wasachieved by capture ELISA (R&D Systems).

6.2 RESULTS

LPS was extracted from Escherichia coli strain W3110 and a htrB1::Tn10,msbB::Ωcam double mutant derivative of W3110, strain MLK986 (Karrow etal., 1992, J. Bacteriol 174:702-710) after culturing them at 30° C., 37°C. or 42° C. as described (Westphal et al., 1965, Met. Carbohyd. Res.5:83-91). To characterize the proinflammatory activity of LPS harvestedfrom W3110 and MLK986 bacilli cultured at over this temperature range,we measured the level of TNFα in culture supernatants 8 hr afterstimulation of human PBMCS. The results showed that significant levelsof TNFα secretion was induced by parent strain W3110 LPS atconcentrations above 1 ng/ml, irrespective of culture temperature (FIG.1). In contrast, LPS from mutant strain MLK986 cultured at 30° C. onlymodestly elicited TNFα secretion and LPS derived from MLK986 bacillicultured at 37° C. and 42° C. even at concentrations as high as 1 μg/mldid not elicit significant TNFα secretion in the human PBMC activationassay (FIG. 1). This observation was reproduced using four unrelatedPBMC donors and two separate LPS preparations. In addition, a similarsecretion pattern was seen for IL-1β in the culture supernatants ofPBMCs after stimulation with each of the MLK986 and W3110 LPSpreparations (Table 1). Secretion of IL-6 was more variable from PBMCdonor to PBMC donor. In some instances LPS from MLK986 cultured eitherat 37° C. or 42° C. induced a modest increase in IL-6 (Table 1).However, the levels were similar to the level of IL-6 induced bynon-pyrogenic synthetic lipid IV_(A) (ICN) and were 5-fold lower thanthe level of IL-6 induced by 100-fold less W3110 LPS (Table 1). Webelieve these data indicate that LPS isolated from MLK986 cultured at orabove 37° C. possesses negligible proinflammatory activity.

TABLE 1 Levels of IL-1β and IL-6 in human PBMC culture supernatantsafter stimulation with LPS LPS source IL-1β IL-6 (Culture Temperature)(pg/ml) (pg/ml) None 12 ± 1 2296 ± 43  W3110 (37° C.) 7032 ± 366 30,698± 120   (10 ng/ml) MLK986 (30° C.) 1918 ± 182 16,000 ± 26   (1 μg/ml)MLK986 (37° C.) 12 ± 3 7593 ± 221 (1 μg/ml) MLK986 (42° C.) 11 ± 4 5164± 144 (1 μg/ml) Lipid IV_(A) (42° C.) 13 ± 1 7581 ± 20  (1 μg/ml)

To investigate whether MLK986 LPS possesses LPS antagonist activity,MLK986 LPS (cultured at 37° C.) at 1 μg/ml was mixed with varyingamounts of W3110 LPS (also cultured at 37° C.) from 1 ng/ml to 100 ng/mland these mixtures were used to stimulate human PBMCs as outlined above.W3110 LPS (also cultured at 37° C.) from 1 ng/ml to 100 ng/ml alone andW3110 LPS (10 ng/ml) mixed with synthetic lipid IV_(A) (1 μg/ml) wereused as controls. The level of TNFα in culture supernatants collected 8hr after stimulation shows that W3110 LPS at a concentration of 1 ng/mland above elicited TNFα secretion but that MLK986 LPS significantlyantagonized this response (Table 2). Interestingly, LPS from MLK986cultured at 42° C. produced did not display LPS antagonist activity.Since culture temperature has been shown to influence LPS aggregationand influence LPS activity (Shnyra et al., 1993, Infection and Immunity61:5351-5360), this later finding may be due to differential aggregationproperties of MLK986 LPS produced under the distinct culture conditions.

TABLE 2 LPS antagonist properties of MLK986 LPS TNFα Treatment (pg/ml)W3110 (100 ng/ml) 1404 ± 40 W3110 (10 ng/ml) 1192 ± 37 W3110 (1 ng/ml) 536 ± 178 MLK986/37° C. (1 μg/ml) <30 MLK986/37° C. (1 μg/ml) + <30W3110 (100 ng/ml) MLK986/37° C. (1 μg/ml) + <30 W3110 (10 ng/ml)MLK986/37° C. (1 μg/ml) + <30 W3110 (1 ng/ml) Lipid IV_(A) (1 μg/ml) +<30 W3110 (10 ng/ml)

In light of the growing importance of β chemokines in microbialinfection (Murphy, 1994, Ann. Rev. Immunol. 12:593-633; Cocchi et al.1955, Science 270:1811-1815), we investigated whether LPS from MLK986 orRSDPLA were capable of eliciting MIP-1α, MIP-1β or RANTES secretion fromhuman PBMCs in vitro. Human PBMCs were stimulated with various dosesMLK986 LPS or RSDPLA (Kitchens et al. 1992, J. Exp. Med. 176:485-494)and the levels of MIP-1α, MIP-1β or RANTES in the culture supernatants8, 16, 24, and 48 hr after stimulation were determined by quantitativecapture ELISA (R&D Systems). Reproducibly, we found that LPS fromMLK986, cultured at either 37° C. or 42° C., and RsDPLA stimulated thesecretion of MIP-1α and MIP-1β in a dose-dependent manner (FIG. 2); thepeak production of these chemokines occurred 24 hr after stimulation(FIG. 3).

6.3 Discussion

In this example, we presented the novel finding that LPS antagonists,both E. coli mutant LPS and R. sphaeroides diphosphoryl lipid A(RsDPLA), possess subtle biological activity in a human PBMC activationassay. The ability of these LPS antagonists to elicit MIP-1α and MIP-1βis inconsistent with a passive competitive inhibition model andtherefore suggests a more complicated mechanism. Irrespective of thebasis for this response, the observation that β chemokines MIP-1α andMIP-1β secretion can be elevated in the absence of endogenous pyrogeniccytokines such as TNFα and IL-1β can open new avenues for noveltherapeutic strategies that exploit this host response.

7. EXAMPLE INHIBITION OF HIV-1 REPLICATION IN HUMAN PBMC-DERIVEDMONOCYTES BY NON-PYROGENIC LPS

The following studies were conducted to demonstrate that non-pyrogenicLPS isolated from E. coli htrB1::Tn10 msbB::Ωcam double mutant MLK986 iscapable of inhibiting HIV replication in human cells.

7.1 Method

Isolation of PBMCS and monocyte-derived macrophages. PBMC healthy donorswere placed in 24 well plates at 3×10⁶ cells/ml in 1 ml of growth medium(GM) (RPMI-1640+10% FBS+10% human serum+Penn/Strep). Monocytes which hadbeen isolated by negative selection with magnetic beads to remove T andB cells, were place in a 25 cm² flask and further purified by adherenceto the flask. After washing, the monocytes were cultured in GM as above.

7.2 Results

Induction of β chemokines. These cultures were then treated with the LPSpreparations as indicated. After 24 hours the supernatants werecollected from each these cultures and the level of β-chemokines andTNF-α and IL-1β were measured by ELISA (R&D Systems). The data shown inTable 3 are representative of 3 different experiments, and indicate thatboth MLK986 and wild type LPS induce comparable levels of β-chemokinesin macrophages and PBMC cultures. However, in contrast to wild type LPS,MLK986 LPS did not induce a measurable TNF-α response.

Inhibition of HIV-1 infection. Monocyte-derived macrophages were treatedwith the LPS preparations (1 μg/ml) and then infected with HIV-1_(Ba-L)at 0.5 ng/ml for 3 hours, washed with PBS 4 times, and cultured ingrowth medium for days. Supernatants were collected at days 4, 7, and 10days post-infection, and tested for the presence of p24 antigen by ELISA(Coulter). Representative data from 3 different experiments (Table 4)show that both MLK986 and wild type LPS potently inhibit HIV-1replication in monocyte-derived macrophages. In addition, pretreatmentof human PBMCs with supernatants collected 24 hr after stimulation ofPBMC with various LPS preparations inhibited HIV-1 infection (Table 5).The data shown in FIG. 4A are representative of 4 different experimentsand demonstrate that MLK986/37 inhibits HIV-1 chemokines. Thus,MLK986/37 induced HIV-1 inhibition in MDM is reversed by addition of amixture of neutralizing antibodies against RANTES, MIP-1α, and MIP-1β(from R&D Systems Inc.; 200 ug/ml each) (FIG. 4B). Further, HIV-1replication inhibition occurred without inducing pyrogenic cytokines(FIG. 4C). These results clearly demonstrate that the LPS antagonistMLK986/37 notently inhibits HIV-1 replication.

TABLE 3 Levels of β-chemokines and TNF-α in PBMC culture supernatantsafter 24 hours of LPS stimulation (1 mg/ml). RANTES MIP-1α MIP-1β TNF-αLPS source ng/ml ng/ml ng/ml ng/ml none 0.212 +/− 0.170 +/− 0.416 +/−<30 .030 .008 .052 LPS wt 2.65 +/− 9.22 +/− 6.76 +/− 1.96 +/− 1.98 1.38.580 .969 MLK986 2.489 +/− 8.84 +/− 6.72 +/− <30 (Prep. P) 1.93 .830.587

TABLE 4 HIV-1 p24 expression in macrophages pretreated with LPS. LPSsource Day 4 Day 7 Day 10 none .340 +/− .095 .890 +/− .153 1.64 +/− .562LPS wt .018 +/− .006   27 +/− .0014 .035 +/− .005 MLK986 (Prep. 02) .027+/− .012 .051 +/− .025  .01 +/− .032

TABLE 5 HIV-1 p24 expression in PBMC pretreated with supernatants fromPBMC stimulated with MLK986, wild type LPS (E. coli, SIGMA) (both at 1mg/ml) or control. LPS source Day 4 Day 7 none .889 +/− .068 2.03 +/−.158 LPS wt .039 +/− .032 .081 +/− .033 MLK986 .094 +/− .067 .134 +/−.038

8. EXAMPLES SYNTHETIC LIPID IV_(A) SUPPRESSES HIV REPLICATION WITHOUTINDUCING MEASURABLE LEVELS OF β CHEMOKINES

The following studies were conducted to demonstrate that non-pyrogenicLPS antagonist lipid IV_(A) is capable of inhibiting HIV replication inhuman cells.

8.1 Methods and Results

To investigate the utility of the aforementioned findings, weinvestigated whether commercially available synthetic lipid IV_(A) (ICNInc.) suppressed the replication of HIV-1_(BAL) in human PBMC-derivedmonocytes. Therefore, PBMCs were obtained as outlined in Section 6, seeabove, and placed into 12 well culture flat-bottom plate and incubatedin CM (see example 1) for 12 days at 37° C. in 5% CO₂. Non-adherentcells were removed and the cells were given fresh media every 2 days.After 6 days, the adherent PBMCs were treated with synthetic lipidIV_(A) at 1000 ng/ml, 100 ng/ml and 10 ng/ml for 24 hours. The treatedcells were subsequently infected with 0.200 ng of HIV-1_(BAL) (Cocchi etal, supra) for 2 hours, washed twice and incubated for a further 19days. Culture supernatants were then collected and the level of p24 inthese supernatants was measured by ELISA (R&D Systems). The results ofthis assay show that lipid IV_(A) suppresses the replication ofHIV-1_(BAL) in a dose-dependent manner (FIG. 5). These results provideclear evidence that synthetic LPS antagonists, such as lipid IV_(A), arecapable of suppressing HIV replication over a wide range ofconcentrations.

9. EXAMPLES NON-PYROGENIC LPS SUPPRESSES HIV REPLICATION WITHOUTDISPLAYING LPS ANTAGONIST ACTIVITY

The following studies were conducted to demonstrate that non-pyrogenicLPS isolated from E. coli htrB1::Tn10 msbB::Ωcam double mutant MLK986cultured at 42° C. is capable of inhibiting HIV replication in humancells. 9.1 Methods and Results

We investigated whether LPS isolated from MLK986 cultured at 42° C.(MLK986/42) suppressed the replication of HIV-1_(BAL) in humanPBMC-derived monocytes. This LPS preparation induces lower levels of βchemokines that LPS isolated from MLK986 cultured at 37° C. and did notdisplay LPS antagonist activity (see above). PBMC-derived monocytes wereobtained as outlined in Section 8, see above. After 6 days, thePBMC-derived monocytes were treated with MLK986/42 at 1000 ng/ml, 100ng/ml and 10 ng/ml for 24 hours. The treated cells were subsequentlyinfected with 0.200 ng of HIV-1_(BAL) (Cocchi et al., supra) for 2hours, washed twice and incubated for a further 19 days. Culturesupernatants were then collected and the level of p24 in thesesupernatants was measured by ELISA (R&D Systems). The results of thisassay show that MLK986/42 suppresses the replication of HIV-1_(BAL) in adose-dependent manner.

This example provides direct evidence that non-pyrogenic LPSpreparations that lack LPS antagonist properties, such as MLK986/42, arecapable of suppressing HIV replication over a wide range ofconcentrations.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An in vitro method of reducing human immunodeficiency virus (HIV-1)replication in monocytes comprising introducing to the monocytes acomposition comprising a variant lipopolysaccharide (LPS) component inan amount effective to reduce HIV replication therein, wherein thevariant LPS is an isolated and purified LPS component obtained from adouble mutant E. coli which is designated E coli htrB1::Tn10 msbB::Ωcamstrain having ATCC Accession No. PTA-2794, wherein the LPS component hasthe following properties: (a) exhibits reduced pyrogenicity relative toa wild-type E. coli lipopolysaccharide; (b) stimulates β chemokinesecretion from the mononuclear cells; (c) reduces secretion of TNF-αproinflammatory relative to a wild-type E. coli lipopolysaccharide; and(d) reduces replication of HIV in the monocytes.
 2. The method of claim1 which further comprises administering to the subject a therapeuticallyeffective amount of a chemokine.
 3. The method of claim 2 which furthercomprises administering to the subject a therapeutically effectiveamount of a chemokine selected from the group consisting of RANTES,MIP-1α and MIP-1β.
 4. The method of claim 1 which further comprisescombining the variant LPS with an anti-viral drug.
 5. The method ofclaim 4 in which the anti-viral drug is selected from one or more of thegroup consisting of AZT, 3TC, ddI, ddC, 3TC, and sequinavir.
 6. Themethod of claim 4 in which the anti-viral drug is selected from aprotease inhibitor or a glycosylation inhibitor.
 7. The method of claim1 further comprising combining the variant LPS with a pharmaceuticallyacceptable carrier.
 8. The method of claim 1 wherein said compositionfurther comprises a wild-type lipopolysaccharide containing structureisolated from a gram-negative microorganism.
 9. An in vitro method ofreducing human immunodeficiency virus (HIV-1) replication in monocytes,comprising administering to mononuclear cells a composition comprising avariant lipopolysaccharide (LPS) in an amount effective to reduce HIVreplication, wherein the variant LPS has been isolated/purified from anE. coli double mutant htrB1::Tn10 msbB::Ωcam strain having ATCCAccession No. PTA-2794, and having the following properties: (a)exhibits reduced pyrogenicity relative to a wild-type E. colilipopolysaccharide; (b) induces secretion of one or more β-chemokinesselected from the group consisting of RANTES, MIP-1α and MIP-1β; (c)reduces secretion of one or more pro-inflammatory cytokines selectedfrom the group consisting of TNFα, IL-1β and IL-6 relative to awild-type E. coli lipopolysaccharide; and (d) reduces HIV replication inmonocytes.
 10. The method of claim 9 further comprising combining thevariant LPS with an anti-viral drug other than a lipopolysaccharidevariant of reduced or absent pyrogenicity.
 11. The method of claim 9further comprising combining the variant LPS with a pharmaceuticallyacceptable carrier.
 12. The method of claim 6, wherein the anti-viraldrug comprises a nucleoside derivative.
 13. The method of claim 9,wherein said composition further comprises a wild-typelipopolysaccharide containing structure isolated from a gram-negativemicroorganism.
 14. The method of claim 9, wherein the compositionfurther comprises a nucleoside derivative.
 15. The method of claim 14,where the nucleoside is selected from the group consisting of a modifiedpurine nucleoside, a modified pyrimidine and halogenated nucleosidederivative.
 16. The method of claim 1 wherein the reduction of HIVreplication is determined by measuring HIV-1 p24 antigen levels.
 17. Themethod of claim 12, wherein the nucleoside is selected from the groupconsisting of a modified purine nucleoside, a modified pyrimidine andhalogenated nucleoside derivative.